Evidence For Free Radical Involvement Research Articles

The neurotoxicity of pyridinium metabolites of haloperidol

Haloperydol is a butyrophenone, typical neuroleptic agent characterized as a high antipsychotics effects in the treatment of schizophrenia and in palliative care to alleviation many syndromes, such as naursea, vomiting and delirium. Clinical problems occurs during and after administration of the drug are side effects, particularly extrapyrramidal symptoms (EPS). The neurotoxicity of haloperydol may be initiated by the cationic metabolites of haloperydol, HPP+, RHPP+, formed by oxidation and reduction pathways. These metabolites are transported by human organic cation transporters (hOCT) to several brain structures for exapmle, in substantia nigra, striatum, caudate nucleus, hippocampus. After reaching the dopaminergic neurons inhibits mitochondrial complex I, evidence for free radical involvement, thus leading to neurodegeneration.

Comparison of protein carbonyl and antioxidant levels in brain tissue from intracerebral haemorrhage and control cases

Background: In an attempt to develop a clearer understanding of the pathological mechanisms underlying intracerebral haemorrhage (ICH), the objective of this investigation was to obtain evidence for free radical-induced oxidative damage in brain tissue following intracerebral haemorrhage. Methods: Brain cortex samples were obtained from the ischaemic penumbra (overlying the haematoma) from 10 patients with spontaneous ICH and from six control cases (normal tissue obtained during tumour removal or aneurysm repair). Following extraction via homogenization and subsequent derivatization with dinitrophenylhydrazine (DNPH), tissue samples were analysed for the presence of protein carbonyl moieties (a hallmark of tissue protein oxidation). This procedure involved SDS-polyacrylamide gel electrophoresis and Western immunoblotting using a commercially available primary monoclonal antibody to DNPH, with final visualization of oxidized protein bands via enhanced chemiluminescence. Results: Samples from ICH cases showed a number (10–15) of well-defined bands of medium to strong staining intensity (not present in nonderivatized samples), corresponding to proteins of molecular mass 25–200 kDa, indicating the presence of oxidatively damaged proteins in these samples. However, tissue samples from control cases also showed the presence of oxidized protein bands, with fractionation patterns for individual ICH or control samples being qualitatively and quantitatively similar. In addition, there was no significant difference in the levels of the following antioxidants (as additional indirect markers of free radical activity) in ICH or control brain tissue: glutathione, glutathione peroxidase, glutathione reductase, catalase, superoxide dismutase, and total antioxidant status. Conclusions: It is concluded from the above data that (i) evidence for free radical involvement in ICH based on protein carbonyl analysis should be interpreted with caution, since normal brain tissue contains a surprisingly high proportion of oxidized proteins; (ii) since there is no evidence for increased protein oxidative damage or decreased tissue antioxidant levels in ICH, therapeutic strategies aimed at salvage of potentially viable tissue would not benefit from inclusion of protein protecting antioxidants.

Vitamin E supplementation and endurance exercise: are there benefits?

It has been widely noted that vitamin E shows numerous beneficial effects through and beyond its antioxidative properties; consequently, vitamin E is expected to prevent degenerative diseases. In the field of sports medicine, many studies dealing with vitamin E have been conducted originally from the point of view of its effects on physical performance. Although some earlier studies indicated that vitamin E supplementation could improve physical performance, defects in the study design or statistical analysis were pointed out at a later time. The majority of subsequent well controlled studies have reported no significant effect on physical performance from vitamin E supplementation. Recent studies suggest that endurance exercise may promote free radical generation in the body, and vitamin E may play an important role in preventing the free radical damage associated with endurance exercise. Although there is evidence of free radical involvement in exercise-induced muscle injury, vitamin E supplementation might not be expected to prevent muscle damage caused by exercise in humans without a vitamin E deficiency. Since it is still unclear whether exercise induces lipid peroxidation in the human body, the beneficial effect of vitamin E supplementation on exercise-induced lipid peroxidation has not yet been established. However, it is proposed that as a result of exercise vitamin E may be mobilised from store tissues and redistributed in the body to prevent oxidative damage. Therefore, we are convinced that vitamin E contributes to preventing exercise-induced lipid peroxidation. It has also been indicated that strenuous endurance exercise may enhance the production of oxidised low density lipoprotein (LDL), which plays a key role in the initiation and progression of atherosclerosis. It is also suggested that this enhanced production of oxidised LDL could be reduced if a higher vitamin E status is maintained. Supplementation with 100 to 200mg of vitamin E daily can be recommended for all endurance athletes to prevent exercise-induced oxidative damage and to reap the full health benefits of exercise.

Pharmacologic properties of phenyl N-tert-butylnitrone.

Phenyl N-tert-butylnitrone (PBN) is the parent of a family of nitrones used as spin-trapping agents to trap free radicals. PBN's pharmacological effects in animal models are extensive, ranging from protection against death after endotoxin shock, protection from ischemia-reperfusion injury, to increasing the life span of mice. Recent additions to the list include protection from bacterial meningitis, thalidomide-induced teratogenicity, drug-induced diabetogenesis, and choline-deficient hepatocarcinogenesis. Because PBN reacts with oxygen radicals to produce less reactive species, it has been suggested that this is the basis of its pharmacological effects. However, there has been no hard evidence for this notation. Nevertheless, many investigators have used the presence of PBN's pharmacologic effect as evidence for free radical involvement in their models. Mechanistic studies on the PBN's antisepsis action revealed that PBN inhibits expression of various pro-inflammatory genes, suggesting that the protective action involves more than a straightforward free radical-scavenging mechanism. Previous and recent developments in the investigations on the pharmacologic properties of PBN are described in this review.

Oxidative stress and amyloidosis.

From recent findings concerning free radical injury in several types of amyloidosis, it appears that free radical injury is involved either amyloid formation or in post-fibrillar modification. As we show in this review, among the more than 20 different types of amyloidosis, only a few types of amyloidosis present direct evidence of free radical involvement in amyloid formation. However, if we search further for other types of amyloidosis, other important information on free radical injury may be forthcoming. Free radical injury is probably central for the toxic properties of amyloid deposits.

Metals and Oxidative Damage in Neurological Disorders

Metals and Oxidative Damage in Neurological Disorders

In vivo evidence for free radical involvement in the degeneration of rat brain 5-HT following administration of MDMA ('ecstasy') and p-chloroamphetamine but not the degeneration following fenfluramine.

1. Administration of 3,4-methylenedioxymethamphetamine (MDMA or 'ecstasy') to several species results in a long lasting neurotoxic degeneration of 5-hydroxytryptaminergic neurones in several regions of the brain. We have now investigated whether this degeneration is likely to be the result of free radical-induced damage. 2. Free radical formation can be assessed by measuring the formation of 2,3- and 2,5-dihydroxybenzoic acid (2,3-DHBA and 2,5-DHBA) from salicylic acid. An existing method involving implantation of a probe into the hippocampus and in vivo microdialysis was modified and validated. 3. Administration of MDMA (15 mg kg-1, i.p.) to Dark Agouti (DA) rats increased the formation of 2,3-DHBA (but not 2,5-DHBA) for at least 6 h. Seven days after this dose of MDMA, the concentration of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) was reduced by over 50% in hippocampus, cortex and striatum, reflecting neurotoxic damage. There was no change in the concentration of dopamine or 3,4-dihydroxyphenylacetic acid (DOPAC) in the striatum. 4. p-Chloroamphetamine (PCA), another compound which produces a neurotoxic loss of cerebral 5-HT content, when given at a dose of 5 mg kg-1 also significantly increased the formation of 2.3-DHBA (but not 2,5-DHBA) in the dialysate for over 4.5 h. post-injection starting 2 h after treatment. 5. In contrast, fenfluramine administration (15 mg kg-1, i.p.) failed to increase the 2,3-DHBA or 2,5-DHBA concentration in the dialysate. A single fenfluramine injection nevertheless also markedly decreased the concentration of 5-HT and 5-HIAA in the hippocampus, cortex and striatum seven days later. 6. When rats pretreated with fenfluramine (15 mg kg-1, i.p.) seven days earlier were given MDMA (15 mg kg-1, i.p.) no increase in 2,3-DHBA was seen in the dialysate from the hippocampal probe. This indicates that the increase in free radical formation following MDMA is occurring in 5-HT neurones which have been damaged by the prior fenfluramine injection. 7. Administration of the free radical scavenging agent alpha-phenyl-N-tert-butyl nitrone (PBN; 120 mg kg-1, i.p.) 10 min before and 120 min after an MDMA (15 mg kg-1, i.p.) injection prevented the acute rise in the 2,3-DHBA concentration in the dialysate and attenuated by 30% the long term damage to hippocampal 5-HT neurones (as indicated by a smaller MDMA-induced decrease in both the concentration of 5-HT and 5-HIAA and also the binding of [3H]-paroxetine). 8. These data indicate that a major mechanism by which MDMA and PCA induce damage to 5-hydroxytryptaminergic neurones in rat brain is by increasing the formation of free radicals. These probably result from the degradation of catechol and quinone metabolites of these substituted amphetamines. In contrast, fenfluramine induces damage by another mechanism not involving free radicals; a proposal supported by some of our earlier indirect studies. 9. We suggest that these different modes of action render untenable the recent suggestion that MDMA will not be neurotoxic in humans because fenfluramine appears safe at clinical doses.

Alpha-tocopherol and ginkgo biloba treatment protects lipid peroxidation during ischemic period in rat groin island skin flaps

Oxygen-derived free radicals have been implicated in the causation of cellular injury during low-flow ischemia and during reperfusion of previously completely ischemic tissue; they are also believed to be the causative factor in the no-reflow phenomena. Modulation of these free radical substances has been suggested as a means of decreasing the amount of tissue loss due to ischemia and subsequent reperfusion. Pretreatment of tissues with a variety of agents has been reported to minimize the production of oxygen radicals and augment tissue survival after an ischemic insult. Further evidence of free radical involvement in skin flap necrosis in a rat groin island skin flap model is presented. In addition, the effects of two different free radical scavengers, alpha tocopherol (20 mg/kg intraperitoneally once daily for a week) and ginkgo biloba (5 mg/kg orally twice a day for a week) have been investigated and compared. Since malonyldialdehyde (MDA) is the end product of lipoperoxidation which occurs in cellular membranes in an ischemic period - dependent manner, MDA levels in tissue homogenates were measured 60, 90, and 120 min after an ischemic insult. MDA levels significantly increased in a time-dependent manner during the ischemic period in the control group. Results from the determination of tissue MDA levels at biopsy sites of radical scavenger treated groups compared with the placebo group showed that the ginkgo biloba-treated rat samples had significantly lower MDA levels than control samples only at the 120 min ischemic period (p<0.01). However, protection of lipoperoxidation in alpha tocopherol-treated rat samples was detected after both the 90 and 12 min ischemic periods (p<0.01), and the magnitude of these decreased MDA levels in alpha tocopherol-treated samples was found to be greater than it was after ginkgo biloba treatment. Decreasing free radicals during reperfusion by using these agents, preferably alpha tocopherol, may be beneficial in modulating the no-reflow phenomenon and subsequent reperfusion injury, and may help to improve tissue salvage.

Poster 189 Evidence for free radical involvement in pressure ulcer development: James C. Donofrio, PhD (University of Kentucky Chandler Medical Center/Kentucky Clinic, Lexington, KY); Richard M. Salcido, MD; Steve B. Fisher, PA-C; John M. Carney, PhD; Robert A. Floyd, PhD

Poster 189 Evidence for free radical involvement in pressure ulcer development: James C. Donofrio, PhD (University of Kentucky Chandler Medical Center/Kentucky Clinic, Lexington, KY); Richard M. Salcido, MD; Steve B. Fisher, PA-C; John M. Carney, PhD; Robert A. Floyd, PhD

Endogenous antioxidant changes in the myocardium in response to acute and chronic stress conditions.

Oxygen is a diradical and because of its unique electronic configuration, it has the potential to form strong oxidants (e.g. superoxide radical, hydrogen peroxide and hydroxyl radical) called oxygen free radicals or partially reduced forms of oxygen (PRFO). These highly reactive oxygen species can cause cellular injury by oxidizing lipids and proteins as well as by causing strand breaks in nucleic acids. PRFO are produced in the cell during normal redox reactions including respiration and there are various antioxidants in the cell which scavenge these radicals. Thus in order to maintain a normal cell structure and function, a proper balance between free radical production and antioxidant levels is absolutely essential. Production of PRFO in the myocardium is increased during various in vivo as well as in vitro pathological conditions and these toxic radicals are responsible for causing functional, biochemical and ultrastructural changes in cardiac myocytes. Indirect evidence of free radical involvement in myocardial injury is provided by studies in which protection against these alterations is seen in the presence of exogenous administration of antioxidants. Endogenous myocardial antioxidants have also been reported to change under various physiological as well as pathophysiological conditions. It appears that endogenous antioxidants respond and adjust to different stress conditions and failure of these compensatory changes may also contribute in cardiac dysfunction. Thus endogenous and/or exogenous increase in antioxidants might have a therapeutic potential in various pathological conditions which result from increased free radical production.

Liver damage due to free radicals.

The involvement of free radical reactions in the pathogenesis of liver injury has been investigated for many years in a few defined experimental systems using carbon tetrachloride, excess iron or ethanol as prooxidant agents. More recently, the hepatotoxicity of several other free radical-generating compounds has been characterised mainly in the rat hepatocyte model. In particular, the mechanisms by which drugs like paracetamol, halothane, paraquat or conditions such as ischemia-reperfusion exert their damaging activity to the liver have mostly been clarified. Since we are not trying to cure diseases occurring only in rats, the likely relevance of free radical reactions also in the genesis and progression of human liver injury has been carefully considered. Increasing evidence of free radical involvement is reported for chronic ethanol intoxication and iron overload, but the most striking proof of a causative role of ree radical chain reactions, namely lipid peroxidation, in the acute lethal damage of the hepatocyte has been obtained so far in ischemic hepatitis.

Irreversible Inhibition of Mitochondrial Complex I by 1‐Methyl‐4‐Phenylpyridinium: Evidence for Free Radical Involvement

Incubation of 10 mM 1-methyl-4-phenylpyridinium (MPP+) with sonicated beef heart mitochondria caused an irreversible time-dependent decrease in NADH-ubiquinone-1 (CoQ1) reductase activity (52% inhibition after 1 h). Inclusion of glutathione, ascorbate, or catalase in the incubation mixture protected the NADH-CoQ1 reductase activity. These results suggest that the interaction of MPP+ with complex I induces free radical generation, which in turn leads to the irreversible inhibition of complex I activity. The generation of free radicals by neurotoxin-induced inhibition of complex I has important implications for our interpretation of the increased oxidative stress observed in Parkinson's disease substantia nigra and for our understanding of the cause(s) of dopaminergic cell death in this disorder.

Oxidative stress as a cause of nigral cell death in Parkinson's disease and incidental Lewy body disease. The Royal Kings and Queens Parkinson's Disease Research Group.

We examine the evidence for free radical involvement and oxidative stress in the pathological process underlying Parkinson's disease, from postmortem brain tissue. The concept of free radical involvement is supported by enhanced basal lipid peroxidation in substantia nigra in patients with Parkinson's disease, demonstrated by increased levels of malondialdehyde and lipid hydroperoxides. The activity of many of the protective mechanisms against oxidative stress does not seem to be significantly altered in the nigra in Parkinson's disease. Thus, activities of catalase and glutathione peroxidase are more or less unchanged, as are concentrations of vitamin C and vitamin E. The activity of mitochondrial superoxide dismutase and the levels of the antioxidant ion zinc are, however, increased, which may reflect oxidative stress in substantia nigra. Levels of reduced glutathione are decreased in nigra in Parkinson's disease; this decrease does not occur in other brain areas or in other neurodegenerative illnesses affecting this brain region (i.e., multiple system atrophy, progressive supranuclear palsy). Altered glutathione metabolism may prevent inactivation of hydrogen peroxide and enhance formation of toxic hydroxyl radicals. In brain material from patients with incidental Lewy body disease (presymptomatic Parkinson's disease), there is no evidence for alterations in iron metabolism and no significant change in mitochondrial complex I function. The levels of reduced glutathione in substantia nigra, however, are reduced to the same extent as in advanced Parkinson's disease. These data suggest that changes in glutathione function are an early component of the pathological process of Parkinson's disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Potential involvement of free radical reactions in ultraviolet light-mediated cutaneous damage.

AbstractFree radicals are chemical species characterized by an odd number of orbital electrons or by pairs of electrons of similar directional spin isolated singly in separate orbitals. Consequently most of these agents are highly reactive and usually exhibit an extremely short half‐life, although due to steric and resonance effects some exceptions occur. Some radicals and their precursors, such as the diradical O2 which exists in the triplet state, represent a critical and essential element of normal metabolism of aerobic organisms where, under normal circumstances, controlled reduction of reactive oxygen species occurs via the cytochrome oxidase or cytochrome P‐450 mixed function monooxygenase systems. In addition to reactive oxygen species, organisms may be subjected to a wide‐range of other free radicals or their precursors, including those of both exogenous and endogenous origin. Elaborate defense mechanisms have evolved to avoid cellular damage from these highly reactive species. Enzymes, such as the superoxide dismutase, the glutathione peroxidase/reductase system, and catalase; interactions with conjugated diene systems such as those found in melanins, carotenoids, and tocopherols; and direct reduction by sulphydryl compounds, phenols, and purines represent but a few of these natural defense systems.Despite a strong rationale for considering free radicals as pathologic agents, progress in implicating these agents, or their reactions, in pathologic processes has been arduous. The fore‐most hurdle to providing definitive evidence for free radical involvement rests with the highly transient nature of these species, hardly reaching measurable levels in vivo and thereby making rigorous testing of the hypothesis extremely difficult. Indeed, free radical damage has been studied, for the most part, by indirect means–usually by measurement of known free radical reaction intermediates and products from which free radical involvement is implied. Nevertheless, free radical formation has been shown to occur in UV‐irradiated skin and a considerable body of circumstantial evidence has been amassed that strongly infers that these agents, or reactions initiated by them, are responsible for at least some of the deleterious effects of UV upon skin.

Deferoxamine increases skin flap survival: additional evidence of free radical involvement in ischaemic flap surgery

This study presents further evidence of free radical involvement in skin flap necrosis in a dorsal rat flap model. Rats receiving deferoxamine, a free radical scavenger and iron chelator had significantly less necrosis (p<0.001) than saline treated rats. In a separate experiment, tissue determinations for malonyldialdehyde (MDA) were consistent with the survival results in showing a significant decrease in MDA in all biopsy sites (p<0.05 or less), indicative of reduced lipoperoxidation in the deferoxamine treated rats.

Toxic properties of arachidonic acid on normal, ischemic and reperfused hearts. Indirect evidence for free radical involvement

We studied the effect of arachidonic acid on function and CPK release of normal, ischemic and reperfused isolated rat hearts. Under control conditions arachidonate (10 μg/ml) produced a transient inotropic effect which gradually reversed during a 90 minute perfusion. Creatinephosphokinase (CPK) release was augmented by arachidonic acid, particularly under high flow (pre-ischemic and reperfusion) conditions. Recovery of contractility following reperfusion of ischemic myocardium was significantly depressed by arachidonic acid. Vitamin E (100 nglml) an antioxidant and free radical scavenger, reduced the enzyme leakage and enhanced recovery of contractility of reperfused myocardium. It also prevented the depression in contractility during control perfusion. Similar protective effects were observed by perfusing the heart with reduced calcium but not by nifedipine; a calcium channel blocker, indomethacin; a prostaglandin synthesis inhibitor or nordihydroguarietic acid; a lipoxygenase inhibitor. Arachidonic acid also inhibited membrane Na +/K +-ATPase although it is unlikely that this property mediated its cardiotoxic influence since it was not prevented by vitamin E. In addition, we observed that arachidonic acid increased the coronary resistance of isolated hearts, probably through enhanced calcium influx as this constriction was reduced by low calcium as well as by nifedipine. Thus, arachidonic acid possesses distinct properties. Its cardiotoxic influence is likely mediated by free radical generation.

ESR and optical absorption evidence for free radical involvement in the photosensitizing action of furocoumarin derivatives and for their singlet oxygen production

Frozen aqueous solutions of thymine and its derivatives were irradiated with visible light ( λ > 320 nm ) in the presence of various furocoumarins. ESR analysis revealed the induction of hydrogen adduct free radicals at C-6 position of thymine, only with those furocoumarin derivatives which show a skin-photosensitizing ability. It has been shown, moreover, that the photocycloaddition of psoralen to thymine, which is responsible for the biological effects of this dye, is inhibited when the induction of free radicals in the thymine moiety has been prevented by electron scavengers. It is suggested that the free radicals observed could be involved in the biological photosensitization. The mechanism of free radical generation and singlet oxygen production by furocoumarins were also investigated.

Evidence for free radical involvement in the hydroxylation of proline: Inhibition by nitro blue tetrazolium

The enzyme, protocollagen proline hydroxylase (PPH), hydroxylates specific proline residues in the collagen sequence to hydroxyproline in the presence of molecular 02, ascorbate and Fe2+ [ 1, 21. There is an absolute requirement for cr-ketoglutarate which undergoes oxidative decarboxylation in stoichio metric proportions to the hydroxyproline formed [3]. The role of Fe2+ and ascorbate in this reaction has not been adequately described. We have recently reported that the Fe2+ activates the enzyme by binding to the apoprotein [4, 51. We describe here, an inhibition of proline hydroxylation by the dye, nitro blue tetrazolium (NBT) which undergoes rapid reduction by scavenging free radicals. The inhibition by NBT is competitive with respect to the enzyme, suggesting that free radicals are involved in the hydroxylation of proline, probably in the reduction of the ferri-enzyme to ferro-enzyme.