Nitrone-based Free Radical Trap Research Articles

HPN-07, a free radical spin trapping agent, protects against functional, cellular and electrophysiological changes in the cochlea induced by acute acoustic trauma.

Oxidative stress is considered a major cause of the structural and functional changes associated with auditory pathologies induced by exposure to acute acoustic trauma AAT). In the present study, we examined the otoprotective effects of 2,4-disulfophenyl-N-tert-butylnitrone (HPN-07), a nitrone-based free radical trap, on the physiological and cellular changes in the auditory system of chinchilla following a six-hour exposure to 4 kHz octave band noise at 105 dB SPL. HPN-07 has been shown to suppress oxidative stress in biological models of a variety of disorders. Our results show that administration of HPN-07 beginning four hours after acoustic trauma accelerated and enhanced auditory/cochlear functional recovery, as measured by auditory brainstem responses (ABR), distortion product otoacoustic emissions (DPOAE), compound action potentials (CAP), and cochlear microphonics (CM). The normally tight correlation between the endocochlear potential (EP) and evoked potentials of CAP and CM were persistently disrupted after noise trauma in untreated animals but returned to homeostatic conditions in HPN-07 treated animals. Histological analyses revealed several therapeutic advantages associated with HPN-07 treatment following AAT, including reductions in inner and outer hair cell loss; reductions in AAT-induced loss of calretinin-positive afferent nerve fibers in the spiral lamina; and reductions in fibrocyte loss within the spiral ligament. These findings support the conclusion that early intervention with HPN-07 following an AAT efficiently blocks the propagative ototoxic effects of oxidative stress, thereby preserving the homeostatic and functional integrity of the cochlea.

Effectiveness of 4-hydroxy phenyl N-tert-butylnitrone (4-OHPBN) alone and in combination with other antioxidant drugs in the treatment of acute acoustic trauma in chinchilla

Acute acoustic trauma (AAT) results in oxidative stress to the cochlea through overproduction of cellular reactive oxygen, nitrogen, and other free radical species appearing from 1 h to 10 days after noise exposure. It has been shown that N-acetyl-L-cysteine (NAC), a glutathione prodrug, and acetyl-L-carnitine (ALCAR), a mitochondrial biogenesis agent, are effective in reducing noise-induced hearing loss. Phenyl N-tert-butylnitrone (PBN), a nitrone-based free radical trap, appears to suppress oxidative stress in a variety of disorders and several biological models. In this study, we tested whether 4-hydroxy PBN (4-OHPBN), a major metabolite of PBN, administered 4 h after noise exposure is effective in treating noise-induced hearing loss and whether a combination of antioxidant drugs (4-OHPBN plus NAC and 4-OHPBN plus NAC plus ALCAR) provides greater efficacy in attenuating AAT since each agent addresses different injury mechanisms. Chinchilla were exposed to a 105 dB octave-band noise centered at 4 kHz for 6 h. 4-OHPBN and combinations of antioxidant drugs were intraperitoneally administered beginning 4 h after noise exposure. Hearing threshold shifts in auditory brainstem responses and missing outer hair cell counts were obtained. 4-OHPBN reduced threshold shifts in a dose-dependent manner while both drug combinations showed greater effects. These results demonstrate that 4-OHPBN and combinations of antioxidants can effectively treat acute acoustic trauma and drug combinations may increase the effectiveness of treatment and decrease the required individual medication dose.

Atypical effect of some spin trapping agents: reversible inhibition of acetylcholinesterase

N- tert-butyl-α-phenylnitrone (PBN), a widely used nitrone-based free radical trap was recently shown to prevent acetylcholinesterase (AChE) inhibitors induced muscle fasciculations and brain seizures while being ineffective against glutamergic or cholinergic receptor agonist induced seizures. In the present study we compared the effects on AChE activity of four free radical spin traps PBN, α-(4-pyridil-1)-N- tert-butyl nitrone (POBN), N- tert-butyl-α-(2-sulfophenyl)-nitrone (S-PBN) and 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO). The kinetics of AChE inhibition were studied in vitro using a spectrophotometric kinetic assay with AChE from rat brain, diaphragm, electric eel and mouse brain. Spin trapping compounds S-PBN and DEPMPO, in concentrations up to 3 mM did not inhibit hydrolysis of ACh, while PBN and POBN inhibited hydrolysis of ACh in a reversible and concentration-dependent manner. Double reciprocal plots of the reaction velocity against varying ACh concentrations at each inhibitor concentration were linear and generally indicated mixed type inhibition. PBN was the most potent inhibitor of mouse AChE with Ki and Ki′ of 0.58 and 2.99 mM, respectively, and the weakest inhibitor of electric eel AChE. In contrast, POBN showed the highest affinity for electric eel enzyme, with Ki and Ki′ values of 1.065 and 3.15 mM, respectively. These findings suggest that the effect of PBN and POBN on AChE activity does not depend on trapping of damaging reactive oxygen and that in addition to their antioxidant action other pharmacological effects of these compounds should be considered when neuroprotective actions of PBN or POBN are investigated.

Nitrone inhibition of age-associated oxidative damage.

The mechanistic basis of the neuroprotective activity of the nitrone-based free radical trap PBN (alpha-phenyl-N-tert-butyl nitrone) has been investigated extensively. Key observations exclude its simple mass action spin trapping of free radicals activity as the key mechanism of action. These include: A) the fact that it protects in experimental stroke even if administered several hours after the event and B) the fact that its chronic low-level administration to old experimental animals reverses their age-enhanced susceptibility to stroke even several days after the last dosage. PBN was found to inhibit gene induction in several models including stroke and an LPS-mediated septic shock model. Stoke causes inducible nitric oxide synthase (iNOS) to be expressed. High levels of nitric oxide and peroxynitrite (formed from nitric oxide), produced by iNOS, is particularly neurotoxic. PBN inhibits iNOS induction. Therefore, it seems that prevention of the formation of neurotoxic products is a rational mechanism of action of PBN in the stroke model. There is strong rationale to consider that there is an enhanced propensity for a "smoldering" neuro-inflammatory state in the old brain. Reversal of this state by PBN may explain its action in preventing age-enhanced stroke susceptibility in old experimental animals. Significant new findings underscore the importance of neuro-inflammatory processes in neuronal death or dysfunction in Alzheimer's disease. Neuro-inflammatory processes implicate enhanced signal transduction processes. Strong evidence for this is the enhanced p38 kinase activation in neurons near plaques and tangles of the Alzheimer's brain in contrast to normal aged-matched control brain which did not show p38 activation. In rat primary astrocytes p38 activation by the pro-inflammatory cytokine IL-1 beta, as well as by H2O2, was significantly suppressed by PBN. Mechanistically it was shown that PBN suppresses the amount of reactive oxygen species (ROS) produced in mitochondrial respiration. Much evidence indicates that ROS are signaling molecules and that they also are involved to maintaining brain phosphatases in an inactive state. We argue that finding a specific high affinity site mechanism for the neuroprotective action of PBN is unlikely based on the complexity of the system reflecting ROS generation and signal transduction processes that have apparently evolved to maintain adaptive responses. The promising pharmacological activity of molecules like PBN is not diminished by this however, for only excessive amounts of ROS is considered detrimental. The action of PBN in suppressing signal transduction processes, most likely by suppressing ROS production in mitochondrial respiration, effectively controls excessive oxidative damage and prevents induction of genes that form neurotoxic products.

Antioxidants, oxidative stress, and degenerative neurological disorders.

Recently, clinical trials of several neurodegenerative diseases have increasingly targeted the evaluation of the effectiveness of various antioxidants. The results so far are encouraging but variable and thus confusing. Rationale for the possible clinical effectiveness of antioxidants in several degenerative conditions has arisen out of the many years of basic science generally showing that reactive oxygen species (ROS) and oxidative damage are important factors in the processes involved. Aging is one of the most significant risk factors for degenerative neurological disorders. Basic science efforts in our laboratory have centered on exploring the role of ROS and oxidative stress in neurodegenerative processes. The present review brings together some of the basic concepts we have learned by following this approach for the last 20 years and specifically the results we have obtained by following up on our serendipitous findings that a nitrone-based free radical trap, alpha-phenyl-tert-butylnitrone (PBN), has neuroprotective activity in several experimental neurodegenerative models. The mechanistic basis of the neuroprotective activity of PBN does not appear to rely on its general free radical trapping or antioxidant activity per se, but its activity in mediating the suppression of genes induced by pro-inflammatory cytokines and other mediators associated with enhanced neuroinflammatory processes. Neuroinflammatory processes, induced in part by pro-inflammatory cytokines, yield enhanced ROS and reactive nitric oxide species (RNS) as well as other unknown components that have neurotoxic properties. Neurotoxic amounts of RNS are formed by the activity of inducible nitric oxide synthase (iNOS). The demonstration of enhanced 3-nitro-tyrosine formation in affected regions of the Alzheimer's brain, in comparison to age-matched controls, reinforces the importance of neuroinflammatory processes. iNOS induction involves activation by phosphorylation of the MAP kinase p38 and can be induced in cultured astrocytes by IL-1beta or H2O2. The action of PBN and N-acetyl cysteine to suppress the activation of p38 was demonstrated in cultured astrocytes. The demonstration of activated p38 in neurons surrounding amyloid plaques in affected regions of the Alzheimer's brain attest to enhanced signal transduction processes in this neurodegenerative condition. The major themes of ROS and RNS formation associated with neuroinflammation processes and the suppression of these processes by antioxidants and PBN continue to yield promising leads for new therapies. Outcomes of clinical trials on antioxidants will become less confusing as more knowledge is amassed on the basic processes involved.

Increased oxidative stress brought on by pro-inflammatory cytokines in neurodegenerative processes and the protective role of nitrone-based free radical traps

Nitrone-based free radical traps (NFTs) have been shown to be protective in several neurodegenerative models. Our research has strongly implicated that: A) several neurodegenerative conditions exhibit increased levels of pro-inflammatory cytokines which consequently result in increased levels of oxidative stress and B) that NFTs act in part by suppressing oxidative stress through suppression of the action of the cytokine cascade. Acquired Immune Deficiency Syndrome (AIDS) dementia complex (ADC) is one of several conditions where the data collected helped to develop these concepts. Novel observations include demonstration that IL-1β acts on cultured brain glia cells to invoke protein nitration and oxidative stress and that low levels of PBN (α-phenyl tertbutyl nitrone) inhibit this effect. We interpret these data as indicating that PBN prevents IL-1β mediated peroxynitrite formation. Additionally, we have found that the AIDS viral envelope protein gp120 upregulates mRNA for the cytokines TNFα and TNFβ in rat neonatal brain, and that PBN prevents this. Western blots of protein extracts showed upregulation of inducible nitric oxide synthase (iNOS) in gp120 treated neonatal rat brains, and that PBN prevented induction of this enzyme as well. These observations underscore the general concept that PBN inhibits the induction of genes which produce neurotoxic products, one of which is peroxynitrite formed by the reaction of nitric oxide with superoxide, and may act also by inhibiting the induction of cytokines which mediate pro-inflammatory conditions in the brain.

Basal Protein Phosphorylation Is Decreased and Phosphatase Activity Increased by an Antioxidant and a Free Radical Trap in Primary Rat Glia

Reversible protein phosphorylation regulates a wide array of cellular functions. Cells respond to cytokines and various stressors via phosphorylation and thus activation of one or more of the mitogen-activated protein kinase (MAPK) pathways. Involvement of these signal transduction pathways has been implicated in numerous pathologies, including inflammation. Using a primary glia cell culture, we show here that the antioxidantN-acetylcysteine (NAC) and the nitrone-based free radical trap, α-phenyl-N-tert-butyl nitrone (PBN), reduce total basal protein phosphorylation in a concentration-dependent manner as assessed by phosphotyrosine analysis and by [γ-32P]ATP transfer radioassay. In addition we show that NAC inhibits H2O2-induced phosphatase inactivation in glia cell lysate. The PBN- and NAC-induced reduction in protein phosphorylation is accompanied by an increase in phosphatase activity, suggesting that PBN and NAC reduce protein phosphorylation by globally augmenting oxidant-sensitive phosphatase activities. These results partly explain why certain antioxidants also possess anti-inflammatory actions.

Redox-sensitive protein phosphatase activity regulates the phosphorylation state of p38 protein kinase in primary astrocyte culture

Reactive oxygen species (ROS) have been implicated as second messengers that activate protein kinase cascades, although the means by which ROS regulate signal transduction remains unclear. In the present study, we show that interleukin 1beta (IL1beta), H2O2, and sorbitol-induced hyperosmolarity mediate a 5- to 10-fold increase in phosphorylation (activation) of the p38 protein kinase in rat primary glial cells as measured by analyses of Western blots using an antibody directed against the dually phosphorylated (active) p38. Additionally, IL1beta was found to elicit H2O2 synthesis in these cells. Concurrent with p38 phosphorylation, all three stimulation paradigms caused an inhibition of protein phosphatase activity. Phenyl-tert-butyl nitrone (PBN), a nitrone-based free radical trap and N-acetyl-cysteine (NAC), a thiol reducing agent, were examined for their effects on the phosphorylation of p38 as well as phosphatase activity. Pretreatment of cells with either PBN or NAC at 1.0 mM suppressed IL1beta H2O2, and sorbitol-mediated activation of p38 and significantly increased phosphatase activity. These data suggest that ROS, particularly H2O2, are used as second messenger substances that activate p38 in part via the transient inactivation of regulatory protein phosphatases.

Protective Action of Nitrone-Based Free Radical Traps against Oxidative Damage to the Central Nervous System

Considerable evidence indicates that oxidative damage to central nervous system (CNS) contributes to the etiology of several neurodegenerative diseases. α-Phenyl- tert -butyl nitrone (PBN), a commonly used nitrone-based free radical trap (NFT), has been shown to protect in several experimental models of neuro-degenerative disease. The actions of PBN are trapping of crucial directly damaging reactive free radicals and rendering them unreactive and preventing the induction of inducible nitric oxide synthase (iNOS) by reactive oxygen species, thus preventing the formation of large tissue levels of nitric oxide (NO). This explains its potent neuroprotective action, especially when it is considered that higher levels of NO are neurotoxic. Many neurodegenerative problems that develop with age appear to be related to increase or altered cytokine changes in brain, known to cause increased NO formation. This implicates that the suppression of iNOS gene expression by PBN and related compounds or the prevention of excess glutamate-mediated NO formation may prove therapeutically useful to protect in a wide range of neurodegenerative diseases. There is discussion on how CNS is sensitive to oxidative damage. Three important points have been put forward: brain is sensitive to oxidative damage, older brain is more sensitive to oxidative damage than younger brain, and age-related lesions in brain mitochondria are related to the increased sensitivity of older brain to oxidative damage. The chapter details the spin-trapping reactions of nitrone-based free radical traps, antioxidant activity of PBN and its protective activity in septic shock models, how PBN and related compounds are neuroprotective, the pharmacology studies of PBN and related compounds, how PBN prevents LPS-mediated NO formation in liver, and the NO chemistry and its potential neurotoxic action. Mentioned is a hypothesis to explain the neuroprotective action of PBN. PBN exhibits neuroprotective action in brain because it prevents the induction of iNOS.

Chapter 13 Nitrone-Based Free Radical Traps as Neuroprotective Agents in Cerebral Ischaemia and Other Pathologies

Nitrone-based spin trapping compounds have been shown to protect experimental animals from pathology associated with ischaemia/reperfusion injury, endotoxaemia, natural and accelerated aging, certain xenobiotics, and physical trauma. Moreover, these compounds have an intriguing nootropic action. Nitrones affect pathophysiological correlates in both the central nervous system and peripheral organ systems. These compounds have been shown to affect cellular oxidation state and oxidatively sensitive enzyme systems, but the precise mode of nitrone action has not been elucidated. Recent discoveries regarding the ability of nitrones to suppress gene transcriptional events associated with pathophysiological states, particularly the elaboration of NF kappa B-regulated cytokines and inducible nitric oxide synthase, argue that nitrones may act at a proximal level to oxidatively sensitive signal amplification systems.