Pulmonary toxicity caused by acute exposure to mercury vapor is enhanced in metallothionein-null mice.

This study examined the protective role of metallothionein (MT) against pulmonary damage caused by acute exposure to metallic mercury (Hg0) vapor using MT-null and wild-type mice. Both strains of mice were exposed to Hg0 at 6.6 to 7.5 mg/m3 for 4 hr each day for 3 consecutive days. This dosing protocol was lethal to over 60% of MT-null mice but did not kill any wild-type mice. More severe pulmonary damage was found by histopathological observation in MT-null mice than in wild-type mice. MT levels in the lung were elevated in wild-type mice after Hg0 vapor exposure, and gel filtration of the lung cytosol revealed that most of the mercury was associated with MT. In MT-null mice, MT levels were below the limit of detection (0.2 microg/g tissue) for the MT assay even after exposure. After exposure to Hg0 vapor for 3 consecutive days, the pulmonary mercury levels in wild-type mice were significantly higher than in MT-null mice. These findings suggest that MT plays a protective role against the acute pulmonary toxicity of Hg0 vapor.     

-phenyl-tert-butyl-nitrone inhibits free radical release in brain concussion

Traumatic brain injury (TBI) is one of the important causes of mortality and morbidity. The pathogenesis of the underlying brain dysfunction is poorly understood. Recent data have suggested that oxygen free radicals play a key role in the primary and secondary processes of acute TBI. We report direct electron spin resonance (ESR) evidence of hydroxyl (·OH) radical generation in closed-head injury of rats. Moderate brain concussion was produced by controlled and reproducible mechanical, fixed, closed-head injury. A cortical cup was placed over one cerebral hemisphere within 20 min of the concussion, perfused with artificial cerebrospinal fluid (aCSF) containing the spin trap agent pyridyl-N-oxide-tert-butyl nitrone (POBN, 100 mM), and superfusate samples collected at 10 min intervals for a duration up to 130 min post brain trauma. In addition, POBN was administered systematically (50 mg/kg body wt.) 10 min pretrauma and 20 min posttrauma to improve our ability to detect free radicals. ESR analysis of the superfusate samples revealed six line spectra (_N = 15.4 and ^β_H = 2.5 G) characteristic of POBN-OH radical adducts, the intensity of which peaked 40 min posttrauma. The signal was undetectable after 120 min. Administration of -phenyl-tert-butyl-nitrone (PBN), a spin adduct forming agent systemically (100 mg/kg body wt. IP 10 min prior to concussion) alone or along with topical PBN (100 mM PBN in aCSF),6significantly (P< 0.001) attenuated the ESR signal, suggesting its possible role in the treatment of TBI.     

Detection of nucleic acid-derived radicals formed by alloxan

Pyrimidine base-derived radical spin adducts were detected in reaction mixtures containing pyrimidine bases, glutathione, and alloxan by the ESR spin trapping technique with a spin trap, alpha-phenyl-N-tert-butyl nitrone (PBN). Pyrimidine nucleoside- and nucleotide-, and ribose- and deoxyribose-derived radical spin adducts of PBN were also observed. However, purine base- and nucleoside-derived radical spin adducts of PBN were not detected. A cytosine-derived radical spin adduct of PBN was not generated under anaerobic conditions. Catalase and mannitol inhibited the formation of the cytosine-derived radical spin adduct of PBN but superoxide dismutase (SOD) did not. EDTA stimulated it and desferrioxamine suppressed it nearly completely. From these results it is presumed that the hydroxyl radical is involved in the formation of the cytosine-derived radical spin adduct of PBN generated by alloxan.     

Protective Effect of Spin Trap Agent,N - tert -butyl-α-phenylnitrone on Hyperoxia-induced Oxidative Stress and Its Potential As a Nitric Oxide Donor

We have previously suggested that the spin trap agent, N - tert -butyl- &#102 -phenylnitrone (PBN) can function not only as an antioxidant but also as a nitric oxide (NO) donor. To characterize the pharmacological activities of PBN against oxidative damage, we examined the effect of PBN on NO generation under hyperoxic conditions. The formation of NO in mice exposed to 95% oxygen was determined using a NOx analyzer and electron spin resonance (ESR). Levels of NOx, an oxidative product of NO, increased in the blood of mice under these conditions. However, the increase was returned to a normal level by the NOS (nitric oxide synthase) inhibitor, L-NMMA, indicating that the NO was formed via a biosynthetic pathway. In addition, ESR spectra of the liver and brain of control and experimental mice that were measured using Fe(DETC) 2 as an NO trap reagent showed strong ESR signals from NO complexes in the livers of mice exposed to 95% oxygen. When examining the effect of PBN in mice, PBN reduced the NOx formation in the blood under the same hyperoxic conditions. In addition, the ESR intensity of the NO complex was weaker in the PBN-treated mice than in the non-treated mice, showing that PBN possess anti-inflammatory properties. However, under a normal atmosphere, NOx and ESR analyses showed that NO levels increased in PBN-treated mice but not in control mice. These findings suggested that PBN functions as an NO donor under specific physiological conditions. PBN appears to protect against hyperoxia-induced NO toxicity by anti-inflammatory action rather than by serving as an NO donor.     

Protective effect of spin trap agent, N-tert-butyl-alpha-phenylnitrone on hyperoxia-induced oxidative stress and its potential as a nitric oxide donor

We have previously suggested that the spin trap agent, N-tert-butyl-alpha-phenylnitrone (PBN) can function not only as an antioxidant but also as a nitric oxide (NO) donor. To characterize the pharmacological activities of PBN against oxidative damage, we examined the effect of PBN on NO generation under hyperoxic conditions. The formation of NO in mice exposed to 95% oxygen was determined using a NOx analyzer and electron spin resonance (ESR). Levels of NOx, an oxidative product of NO, increased in the blood of mice under these conditions. However, the increase was returned to a normal level by the NOS (nitric oxide synthase) inhibitor, L-NMMA, indicating that the NO was formed via a biosynthetic pathway. In addition, ESR spectra of the liver and brain of control and experimental mice that were measured using Fe(DETC)2 as an NO trap reagent showed strong ESR signals from NO complexes in the livers of mice exposed to 95% oxygen. When examining the effect of PBN in mice, PBN reduced the NOx formation in the blood under the same hyperoxic conditions. In addition, the ESR intensity of the NO complex was weaker in the PBN-treated mice than in the non-treated mice, showing that PBN possess anti-inflammatory properties. However, under a normal atmosphere, NOx and ESR analyses showed that NO levels increased in PBN-treated mice but not in control mice. These findings suggested that PBN functions as an NO donor under specific physiological conditions. PBN appears to protect against hyperoxia-induced NO toxicity by anti-inflammatory action rather than by serving as an NO donor.     

Radical Trapping and Inhibition of Iron-Dependent CNS Damage by Cyclic Nitrone Spin Traps

Abstract: Oxidative damage in the CNS is proposed to play a role in many acute and chronic neurodegenerative disorders. Accordingly, the nitrone spin trap α-phenyl- N - tert -butylnitrone (PBN), which reacts covalently with free radicals, has shown efficacy in a variety of animal models of CNS injury. We have synthesized a number of cyclic variants of PBN and examined their activity as radical traps and protectants against oxidative damage in CNS tissue. By using electron spin resonance spectroscopy, the cyclic nitrones MDL 101,002 and MDL 102,832 were shown to trap radicals in a manner similar to that of PBN. All cyclic nitrones tested prevented hydroxyl radical-dependent degradation of 2-deoxyribose and peroxyl radical-dependent oxidation of synaptosomes more potently than PBN. The radical scavenging properties of the cyclic nitrones contributed to a three- to 25-fold increase in potency relative to PBN against oxidative damage and cytotoxicity in cerebellar granule cell cultures. Similar to the phenolic antioxidant MDL 74,722, the nitrones minimized seizures and delayed the time to death in mice following central injection of ferrous iron. Although iron-induced lipid peroxidation was inhibited by MDL 74,722, the nitrones had no effect on this biochemical end point, indicating that iron-induced mortality does not result solely from lipid peroxidation and suggesting additional neuroprotective properties for the nitrones. These results indicate that cyclic nitrones are more potent radical traps and inhibitors of lipid peroxidation in vitro than PBN, and their ability to delay significantly iron-induced mortality in vivo suggests they may be useful in the treatment of acute and chronic neurodegeneration. Furthermore, the stability of the spin trap adducts of the cyclic nitrones provides a new tool for the study of oxidative tissue injury.     

Detection of spin adducts in blood after administration of carbon tetrachloride to rats.

Rats were treated with CCl4 and the spin trapping agent alpha-phenyl-N-t-butyl nitrone (PBN), followed by ESR investigations on samples of heparinized blood. The major signal detected was the ascorbate semidione radical, but smaller concentrations of the carbon dioxide radical anion spin adduct of PBN could also be detected. The ESR signals were more intense when experiments were conducted with plasma, rather than blood. The spin adducts detected were not associated with the red blood cells, and their apparent concentrations increased when the cells were removed by centrifugation. The addition of ascorbate oxidase to the samples markedly diminished the intensity of the ascorbate semidione radical. When plasma samples from CCl4-treated rats were extracted into toluene, the ESR spectrum of the trichloromethyl adduct of PBN was observed in the extract. Because the spectrum of this adduct was not observed in direct ESR studies of plasma, it is possible that immobilization occurred in the presence of plasma proteins. Evidence to support this hypothesis was developed by adding bovine serum albumin (BSA) to an aqueous solution of the trichloromethyl radical adduct of PBN. As the BSA concentration was increased, the intensity of the ESR spectrum was markedly diminished, and displayed features of an immobilized adduct.     

Clarification of the Relationship Between Free Radical Spin Trapping and Carbon Tetrachloride Metabolism in Microsomal Systems

It has been proposed that the C-phenyl-N-tert-butylnitrone/trichloromethyl radical adduct (PBN/^•CCl₃) is metabolized to either the C-phenyl-N-tert-butylnitrone/carbon dioxide anion radical adduct (PBN/^•CO₂⁻) or the glutathione (GSH) and CCl₄-dependent PBN radical adduct (PBN/[GSH-^•CCl₃]). Inclusion of PBN/^•CCl₃ in microsomal incubations containing GSH, nicotinamide adenine dinucleotide phosphate (NADPH), or GSH plus NADPH produced no electron spin resonance (ESR) spectral data indicative of the formation of either the PBN/[GSH-^•CCl₃] or PBN/^•CO₂⁻ radical adducts. Microsomes alone or with GSH had no effect on the PBN/^•CCl₃ radical adduct. Addition of NADPH to a microsomal system containing PBN/^•CCl₃ presumably reduced the radical adduct to its ESR-silent hydroxylamine because no ESR signal was observed. The Folch extract of this system produced an ESR spectrum that was a composite of two radicals, one of which had hyperfine coupling constants identical to those of PBN/^•CCl₃. We conclude that PBN/^•CCl₃ is not metabolized into either PBN/[GSH-^•CCl₃] or PBN/^•CO₂⁻ in microsomal systems.     

Spin trapping the cysteine thiyl radical with phenyl-N-t-butylnitrone

The cysteine thiyl radical has been detected in a variety of biological systems by means of the ESR spectrum of the adduct between the radical and nitrone spin traps. 5,5-Dimethyl-1-pyroline N-oxide (DMPO) is the spin trap of choice in these studies for several reasons. However, we show here that the adduct between the cysteine thiyl radical and phenyl-N-t-butylnitrone (PBN) spin trap can be observed under certain oxidizing conditions where the adduct with DMPO is not detected. This suggests the use of PBN in searching for the thiyl radical under such conditions.     

α-phenyl-tert-butyl-nitrone inhibits free radical release in brain concussion

Traumatic brain injury (TBI) is one of the important causes of mortality and morbidity. The pathogenesis of the underlying brain dysfunction is poorly understood. Recent data have suggested that oxygen free radicals play a key role in the primary and secondary processes of acute TBI. We report direct electron spin resonance (ESR) evidence of hydroxyl (·OH) radical generation in closed-head injury of rats. Moderate brain concussion was produced by controlled and reproducible mechanical, fixed, closed-head injury. A cortical cup was placed over one cerebral hemisphere within 20 min of the concussion, perfused with artificial cerebrospinal fluid (aCSF) containing the spin trap agent pyridyl-N-oxide-tert-butyl nitrone (POBN, 100 mM), and superfusate samples collected at 10 min intervals for a duration up to 130 min post brain trauma. In addition, POBN was administered systematically (50 mg/kg body wt.) 10 min pretrauma and 20 min posttrauma to improve our ability to detect free radicals. ESR analysis of the superfusate samples revealed six line spectra (α_N = 15.4 and α^β_H = 2.5 G) characteristic of POBN-OH radical adducts, the intensity of which peaked 40 min posttrauma. The signal was undetectable after 120 min. Administration of α-phenyl-tert-butyl-nitrone (PBN), a spin adduct forming agent systemically (100 mg/kg body wt. IP 10 min prior to concussion) alone or along with topical PBN (100 mM PBN in aCSF),6significantly (P< 0.001) attenuated the ESR signal, suggesting its possible role in the treatment of TBI.     

Alpha-phenyl-tert-butylnitrone (PBN) inhibits NFkappaB activation offering protection against chemically induced diabetes

Alpha-phenyl-tert-butylnitrone (PBN) is an effective spin trapping agent by reacting with and stabilizing free radical species. Reactive oxygen species (ROS) have been implicated in pancreatic beta cell death and the development of insulin-dependent diabetes mellitus (IDDM). We speculate that treatment with the PBN, will protect against diabetes development in two distinct chemically induced models for IDDM. Pretreatment with PBN (150 mg/kg ip) significantly reduced the severity of hyperglycemia in both alloxan- and streptozotocin (STZ) induced diabetes. To determine the mechanism by which PBN prevents hyperglycemia, we examined the ability of PBN to inhibit NFkappaB activation and to stabilize alloxan- and STZ-induced radicals. Both alloxan and STZ induced NFkappaB activation in the pancreas 30 min after their injection (50 mg/kg iv). PBN pretreatment inhibited both alloxan- and STZ-induced activation of NFkappaB and nitric oxide production. EPR studies showed that PBN could effectively trap alloxan-induced free radicals. It is clear that PBN can inhibit NFkappaB activation in the pancreas and reduce hyperglycemia in two distinct diabetogenic compounds. This research indicates that NFkappaB activation may be a key signal leading to beta cell death and IDDM. Understanding the cellular pathways leading to beta cell death may help in developing effective preventive or therapeutic targets for IDDM.     

Hydroxyl and 1-hydroxyethyl free radical detection using spin traps followed by derivatization and gas chromatography—mass spectrometry

Summary

Detection of hydroxyl free radicals is frequently performed by electron spin resonance (ESR) following spin trapping of the radical using 5,5-dimethylpyrroline N-oxide (DMPO) to generate a stable free radical having a characteristic ESR spectrum. The necessary ESR equipment is expensive and not readily available to many laboratories. In the present study, a specific and sensitive gas chromatography—mass spectrometry (GC/MS) method for detection of hydroxyl and hydroxyethyl free radicals is described. The DMPO or N-t-butyl—α—phenylnitrone (PBN) radical adducts are extracted and derivatized by trimethylsylilation and analyzed by GC/MS. To standardize the method, ^.OH and 1-hydroxyethyl radicals were generated in two different systems: 1) a Fenton reaction in a pure chemical system in the absence or presence of ethanol and 2) in liver microsomal suspensions where ethanol is metabolized in the presence of NADPH. In the Fenton system both radicals were easily detected and specifically identified using DMPO or PBN. In microsomal suspensions DMPO proved better for detection of ^.OH radicals and PBN more suitable for detection of 1-hydroxyethyl radicals. The procedure is specific, sensitive and potentially as useful as ESR.     

Structure Identification of Free Radicals by Esr And Gc/Ms of Pbn Spin Adducts From the In Vitro and in vivo Rat Liver Metabolism Of Halothane

Free radicals were detected from the in vim metabolism of halothane (rat liver microsomes) by the PBN spin trapping method. The detected radical species include the I-chloro-2.2.2-trifluoro-I-ethyl radical (I). as determined by mass spectral analysis, and lipid-type radicals assigned by high resolution ESR spectro-scopy with the use of d, deuterated PBN. The lipid-derived radicals are a carbon-centred radical with the partially assigned structure 'CH, R and an oxygen-centred radical of the OR type. From the mass spectral analysis of the spin adduct mixture there is also evidence for a halocarbon double adduct of PBN of the type I-PBN-I.     

Spin trapping evidence for the lack of significant hydroxyl radical production during the respiration burst of human phagocytes using a spin adduct resistant to superoxide-mediated destruction

Failure to detect hydroxyl radical (.OH)-derived spin adducts of 5,5-dimethyl-1-pyrroline N-oxide in electron spin resonance (ESR) spin trapping experiments has been offered as evidence for the lack of the endogenous capacity of stimulated human phagocytes (neutrophils, monocytes, and monocyte-derived macrophages (MDM] to generate .OH. Recent reports that 5,5-dimethyl-1-pyrroline N-oxide spin adducts are unstable in the presence of superoxide-generating systems such as stimulated neutrophils has raised concerns regarding the sensitivity of spin trapping techniques for assessment of phagocyte free radical formation. Consequently, we have employed a new approach that uses the spin trap N-t-butyl-alpha-phenyl-nitrone (PBN) and dimethyl sulfoxide. In the presence of dimethyl sulfoxide and PBN, the formation of .OH via three different mechanisms in air-saturated aqueous solutions all yielded a single nitroxide species whose ESR peak amplitude remained stable in the presence of superoxide (.O2-). This nitroxide, which we have assigned as PBN/.OCH3, appears to be an oxygen-centered radical derived from the spin trapping of the reaction product of O2 and methyl radical. When neutrophils, monocytes, or MDM were stimulated with phorbol 12-myristate 13-acetate or opsonized zymosan in the presence of exogenous iron, catalase-inhibitable PBN/.OCH3 was the sole nitroxide detected. In the absence of exogenous iron, no nitroxide was observed, providing evidence for the lack of the endogenous capacity of neutrophils, monocytes, and MDM to generate .OH.     

Cadmium induced limb defects in mice: strain associated differences in sensitivity.

Cadmium (CdSO4) was given ip on day 9 at 12 or 24 mumol/kg to pregnant CD-1 (non-inbred) mice. Fetuses showed malformations of the limbs, face, trunk, and tail. There was a statistically significant relationship between the dose of cadmium and the malformation rate. Cadmium (12 mumol/kg ip on day 9) was then given to mice of six inbred strains three of which (A/J, BALB/cJ, and C57BL6J) carry a gene cdm for resistance to cadmium-induced testicular damage, and three strains (AKR/J, CBA/J, and DBA/2J) which do not. Paradoxically, the three strains resistant to cadmium induced testicular damage were significantly more sensitive to its teratogenic effects than were the other three strains. In all inbred strains most malformations involved the limbs. All forelimb defects found in inbred or non-inbred cadmium treated mice were postaxial and indistinguishable from those produced by acetazolamide in mice. The remarkable similarity of the cadmium- and acetazolamide-induced forelimb malformations may be a reflection of the limited number of ways that a rodent forelimb can react to a teratogenic insult. The hindlimb defects were all preaxial.     

In vivo detection of free radicals induced by diethylnitrosamine in rat liver tissue

Diethylnitrosamine (DEN) is a well-known carcinogenic substance that requires microsomal activation before it can react with DNA to cause mutations and cancer. The aim of this study was to use in vivo spin trapping and spin probe techniques to investigate whether free radicals are generated in rat liver tissue during DEN activation. We used alpha-phenyl-n-tert-butylnitrone (PBN) as the spin trapping agent, which was delivered through an intraperitoneal injection before DEN administration. One hour after DEN administration, multicomponent PBN adducts in the bile were detected, and the intensities were diminished by the cytochrome P450 inhibitor SKF-525A. A computer simulation of the ESR signals revealed the presence of a lipid-derived radical. Using the in vivo spin probe/ESR technique, the signal decay rate of methoxycarbonyl-PROXYL was significantly increased in the DEN-treated group compared with the rate in the vehicle group. The enhanced signal decay rate was restored with PBN and/or SKF-525A pretreatment. These results suggested that lipid-derived free radicals were generated in the liver within 1 h after DEN administration.     

Involvement of free radicals in cerebral vascular reperfusion injury evaluated in a transient focal cerebral ischemia model of rat

Free radicals have been suggested to be largely involved in the genesis of ischemic brain damage, as shown in the protective effects of alpha-phenyl-N-tert-butyl nitrone (PBN), a spin trapping agent, against ischemic cerebral injury. In the present study, the effects of PBN as well as MCI-186, a newly-developed free radical scavenger, and oxypurinol, an inhibitor of xanthine oxidase, were evaluated in a rat transient middle cerebral aretery (MCA) occlusion model to clarify the possible role of free radicals in the reperfusion injury of brain. The volume of cerebral infarction, induced by 2-h occlusion and subsequent 2-h reperfusion of MCA in Fisher-344 rats, was evaluated. The administration of PBN (100 mg/kg) and MCI-186 (100 mg/kg) just before reperfusion of MCA significantly reduced the infarction volume. In contrast, oxypurinol (100 mg/kg) failed to show any preventive effect on the infarction. These results suggest that free radical formation is involved in the cerebral damage induced by ischemia-reperfusion of MCA, and that hydroxyl radical is responsible for the reperfusion injury after transient focal brain ischemia. It is also suggested that xanthine oxidase is not a major source of free radicals.     

Investigation on the free radical producing effect of hematoporphyrin (a spin trapping study)

Direct evidence was given concerning the free radical production of hematoporphyrin (HP) and its derivative, during illumination, by ESR spectroscopy. A spin trapping measurement was carried out using PBN spin trap for determination of this radical(s). Three different spin-adducts were found. Two of them are likely to be connected to the reactive free radical production of HP.