Increased stress response and beta-phenylethylamine in MAOB-deficient mice.

MAOA and MAOB are key iso-enzymes that degrade biogenic and dietary amines. MAOA preferentially oxidizes serotonin (5-hydroxytryptamine, or 5-HT) and norepinephrine (NE), whereas MAOB preferentially oxidizes beta-phenylethylamine (PEA). Both forms can oxidize dopamine (DA). A mutation in MAOA results in a clinical phenotype characterized by borderline mental retardation and impaired impulse control. X-chromosomal deletions which include MAOB were found in patients suffering from atypical Norrie's disease, which is characterized by blindness and impaired hearing. Reduced MAOB activity has been found in type-II alcoholism and in cigarette smokers. Because most alcoholics smoke, the effects of alcohol on MAOB activity remain to be determined. Here we show that targetted inactivation of MAOB in mice increases levels of PEA but not those of 5-HT, NE and DA, demonstrating a primary role for MAOB in the metabolism of PEA. PEA has been implicated in modulating mood and affect. Indeed, MAOB-deficient mice showed an increased reactivity to stress. In addition, mutant mice were resistant to the neurodegenerative effects of MPTP, a toxin that induces a condition reminiscent of Parkinson's disease.

Oxidative changes in brain pyridine nucleotides and neuroprotection using nicotinamide.

Pyridine nucleotides are critical during oxidative stress due to their roles in reductive reactions and energetics. The aim of the present study was to examine pyridine nucleotide changes in six brain regions of mice after an intracerebroventricular injection of the oxidative stress inducing agent, t-butyl hydroperoxide (t-BuOOH). A secondary aim was to investigate the correlation between NAD+ levels and DNA fragmentation. Here, we demonstrate that t-BuOOH induced a rapid oxidation of NADPH and a slow depletion of NAD+ in most brain regions. A slight increase in NADH also occurred in five brain regions. NAD+ depletion was associated with increased DNA fragmentation. This suggests the initiation of a death cascade involving poly(ADP-ribose) polymerase (PARP), NAD+, ATP depletion and consequent cell death in brain tissue. PARP activity was accelerated in some brain regions after 20 min of oxidative stress. To counteract oxidative stress induced toxicity, NAD+ levels were increased in the brain using an intraperitoneal injection of nicotinamide. A surplus of brain NAD+ prevented DNA fragmentation in some brain regions. Nicotinamide administration also resulted in higher brain NADH, NADP+ and NADPH levels in some regions. Their synthesis was further upregulated during oxidative stress. Nicotinamide as a precursor for NAD+ may provide a useful therapeutic strategy in the treatment of neurodegeneration.

Acrolein-induced oxygen radical formation.

The mechanism of acrolein-induced lipid peroxidation is unknown. This study found that acrolein and its glutathione adduct, glutathionylpropionaldehyde, induce oxygen radical formation. These oxygen radicals may be responsible for the induction of lipid peroxidation by acrolein. The enzymes xanthine oxidase and aldehyde dehydrogenase were found to interact with glutathionylpropionaldehyde to produce O2.- and HO(.). Acrolein was oxidized by xanthine oxidase to produce acroleinyl radical and O2(.-). Aldehyde dehydrogenase metabolized acrolein to form O2.- but not acroleinyl radical. The fact that glutathionylpropionaldehyde is a more potent stimulator of oxygen radical formation than acrolein indicates that glutathionylpropionaldehyde is a toxic metabolite of acrolein and may be responsible for some of the in vivo toxicity of acrolein.

MPP+ and MPDP+ induced oxygen radical formation with mitochondrial enzymes.

MPP+ has been reported to inhibit reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase in mitochondria, which results in the formation of O2(.-). The current report demonstrates that H2O2 and HO. are also products of MPP+ interaction with NADH dehydrogenase. It is possible that MPP. formation precedes the formation of some of these active oxygen species. Reducing equivalents for radical formation come from NADH. MPP+ may be capable of interacting with submitochondrial particles at a site other than the rotenone site, which results in some formation of oxygen radicals. Plasma amine oxidase incubations with MPDP+ resulted in O2.- H2O2, and perhaps HO. formation. This is probably due to MPP. formation from the oxidation of MPDP+. This study presents new findings that indicate the potential importance of oxygen radical formation in mitochondria during MPTP toxicity.

Redox cycling of MPP+: evidence for a new mechanism involving hydride transfer with xanthine oxidase, aldehyde dehydrogenase, and lipoamide dehydrogenase.

MPP+ is redox active in the presence of cytochrome P450 reductase and induces the formation of O2.- and HO(.). In this study, we report the redox cycling capability of MPP+ with additional enzymes and with UV photolysis detected through ESR techniques. The treatment of MPP+ with UV light resulted in the production of HO. trapped as a spin adduct. Two of the enzymes examined in this study, xanthine oxidase and aldehyde dehydrogenase, produced O2.- in the presence of substrate. However, when MPP+ was added to the incubations, the radical trapped by DMPO was HO(.). This indicates that MPP+ redox cycles in the presence of these two enzymes or UV light, which produces HO.. Our data also suggest that MPP+ is reduced by lipoamide dehydrogenase. MPP+ stimulated the oxidation of reduced nicotinamide adenine dinucleotide (NADH) by the enzyme at concentrations between 2 mM and 8 mM of MPP+. Higher concentrations of MPP+ inhibited lipoamide dehydrogenase. MPP+ appears to be redox active with a number of redox enzymes. The mechanism involved may be hydride transfer from the enzymes to MPP+, rather than a direct single-electron reduction.

New aspects of brain oxidative stress induced by tert-butylhydroperoxide.

Many diseases and aging may be associated with oxidative stress in the brain. However, the effects of oxidative stress in the brain should be more clearly described, especially in terms of effects on brain reduced glutathione (GSH). This issue was addressed by intracerebroventricular injection of a direct-acting oxidative stress inducing agent, tert-butylhydroperoxide. Oxidized glutathione (GSSG) levels in the brain increased by as much as 90-fold during tert-butylhydroperoxide-induced oxidative stress. At the same time, brain GSH levels decreased. The brain appears to retain GSSG and not reduce it or export it efficiently. Vitamin E levels in the striatum increased during tert-butylhydroperoxide-induced oxidative stress. Aging alters the ability of the brain to detoxify an oxidative stress, in that 8-month-old mice retain GSSG in their brains much more than 2-month-old mice. Eight-month-old mice were much more susceptible to tert-butylhydroperoxide-induced toxicity than 2-month-old mice. This may indicate that aging makes the brain more susceptible to oxidative damage.

Parkinson's disease--redox mechanisms.

Parkinson's disease occurs in 1% of people over the age of 65 when about 60% of the dopaminergic neurons in the substantia nigra of the midbrain are lost. Dopaminergic neurons appear to die by a process of apoptosis that is induced by oxidative stress. Oxygen radicals abstract hydrogen from DNA forming DNA radicals that lead to DNA fragmentation, activation of DNA protective mechanisms, NAD depletion and apoptosis. Oxygen radicals can be formed in dopaminergic neurons by redox cycling of MPP+, the active metabolite of MPTP. This redox cycling mechanism involves the reduction of MPP+ by a number of enzymes, especially flavin containing enzymes, some of which are found in mitochondria. Tyrosine hydroxylase is present in all dopaminergic neurons and is responsible for the synthesis of dopamine. However, tyrosine hydroxylase can form oxygen radicals in a redox mechanism involving its cofactor, tetrahydrobiopterin. Dopamine may be oxidized by monoamine oxidase to form oxygen radicals and 3,4-dihydroxyphenylacetaldehyde. This aldehyde may be oxidized by aldehyde dehydrogenase with the formation of oxygen radicals and 3,4-dihydroxyphenylacetic acid. The redox mechanisms of oxygen radical formation by MPTP, tyrosine hydroxylase, monoamine oxidase and aldehyde dehydrogenase will be discussed. Possible clinical applications of these mechanisms will be briefly presented.

Recent developments on the role of mitochondria in poly(ADP-ribose) polymerase inhibition.

Numerous pathophysiological disorders involve some element of oxidative stress and bioenergetic deficit. Poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors have been used recently as a promising new therapeutic strategy aimed at halting the bioenergetic decline associated with oxidative brain insults and other conditions. PARP-1 uses NAD+ as a substrate and is activated during stressful circumstances, mainly in the nucleus. PARP-1 inhibitors are well known for blocking the excessive consumption of NAD+, thereby preserving energy metabolism. But what is the role of mitochondria in this process? Recent investigations have begun to focus on whether mitochondrial function can also be preserved by PARP-1 inhibitors. This review will present some of the latest mechanistic evidence documenting the potential involvement of PARP-1 inhibitors in protecting mitochondrial function and preventing necrosis, apoptosis and mitochondrial calcium cycling.

Brain oxidative stress--analytical chemistry and thermodynamics of glutathione and NADPH.

Oxidative stress occurs in the brain due to stroke, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, trauma, aging and other conditions. Analysis of the effects of oxidative stress can involve quantitation of brain GSH, GSSG, NADPH and NADP. Reliable and rapid assays have been developed for these compounds and will be presented in detail. The assays have been used to analyze the effects of brain oxidative stress. Thermodynamic calculations can be performed to find the observed electrochemical potentials of the GSSG/GSH and the NADP/NADPH couples during oxidative stress. The biochemical consequences of these thermodynamic changes in the cell will be discussed as well as the defense mechanisms available to the cell to recover from oxidative stress.

Effects of medium conditioned by retinal pigmented epithelial cells on neurotransmitter phenotype in retinoblastoma cells.

We previously reported that medium conditioned by retinal pigmented epithelial cells can induce cellular differentiation in human retinoblastoma cells. Extensive neurite outgrowth, increased expression of neuronal marker molecules and decreases expression of glial marker molecules are characteristic of the differentiated phenotype. In the studies described here, we examine whether modulations in the expression of potential neurotransmitter molecules, catecholamines and indolealkyl amines, might be associated with the differentiation of retinoblastoma cells. Concentrations of serotonin, 5-hydroxyindoleacetic acid, 3-methoxytyrosine, homovanillic acid, and 3-methoxy-4-hydroxyphenylacetic acid in extracts of differentiated and undifferentiated retinoblastoma cells were assessed by HPLC. The results show that serotonin and its metabolite, 5-hydroxyindoleacetic acid, are characteristically present in undifferentiated cells. Dopa metabolites, 3-methoxytyrosine, homovanillic acid and 3-methoxy-4-hydroxy-phenylacetic acid, are uniquely present in differentiated cells. It appears that differentiation of retinoblastoma cells induced by factors secreted by retinal pigmented epithelial cells involves a switch from a serotonergic phenotype to one dominated by metabolites of dopa. These findings may provide clues about the factors that control retinoblastoma cells and metastasis.

Medicinal chemistry of nicotinamide in the treatment of ischemia and reperfusion.

Nicotinamide can facilitate DNA repair by inhibiting poly(ADP-ribose) polymerase, increasing NAD levels and adjusting other related enzyme activities. This review will summarize recent work on the design of poly(ADP-ribose) polymerase inhibitors, poly(ADP-ribose) glycohydrolase inhibitors and will discuss the possible use of drugs that interact with NAD synthetic enzymes.

Apoptosis and oxidative stress in the aging brain.

DNA is a primary site of damage during oxidative stress in the brain. DNA fragmentation occurs within minutes of induction of oxidative stress. This DNA fragmentation probably results from the attack of free radicals on DNA and from the activation of endonucleases. Oxidative stress was induced by intracerebroventricular injection of t-butylhydroperoxide. This results in a very rapid flux of t-butylhydroperoxide, which is cleared from the brain within minutes. This flux of t-butylhydroperoxide results in the formation of hydroxyl radical in the brain and probably in the nuclei of brain cells. Necrosis results from extensive DNA fragmentation caused by massive oxidative stress. Cresyl violet stained brain sections demonstrated necrosis in many brain regions. In addition, previous electron microscopy studies showed degradation of cellular nuclei caused by tBuOOH toxicity. Low doses of t-butylhydroperoxide can induce apoptosis, which is a delayed form of cell death. Apoptosis was found in brains stained to visualize apoptotic DNA fragments. Experiments performed in mice aged 2, 8 or 24 months will be discussed. We have also found that apoptosis and DNA fragmentation can be prevented by pretreating mice with the vitamin micotinamide. Nicotinamide is a precursor for NAD. DNA repair requires high levels of NAD in the nucleus for the activity of poly(ADP-ribose) polymerase. Oxidative stress in the brain produces both necrosis and apoptosis, probably as the result of DNA fragmentation. Senescence is associated with an increase in the production of DNA fragments during brain oxidative stress, which probably leads to more necrosis and apoptosis than in younger mice.

Effects of MPTP on the cerebrovasculature.

The neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, has been shown to cause pooling of blood in the brain microvasculature and decrease the permeability of the blood-brain barrier. All areas of the brain examined in this study were affected. This study points out the possibility that reduced nutrient uptake, hypoxia and ensuring free radical damage are involved in the mechanism of toxicity of this neurotoxin.

Increased brain NAD prevents neuronal apoptosis in vivo.

Apoptosis is a characteristic form of cell death which has been implicated in neurodegeneration. In this study we document the induction of apoptosis and DNA fragmentation in vivo by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin. MPTP selectively damages dopaminergic neurons in the substantia nigra of the midbrain. It is a potent inducer of oxygen radicals. Nicotinamide, a precursor of NAD, is able to block the apoptosis induced by MPTP. Nicotinamide also quenches some of the radicals formed by xanthine oxidase. Nicotinamide may be of interest in the treatment of neurodegeneration.

MPTP toxicity in the mouse brain and vitamin E.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) caused transient alterations in vitamin E levels in every brain region examined. However, vitamin E returned to normal levels within a few hours in all brain regions but the substantia nigra, where at 2 days vitamin E levels first rose above normal levels. Vitamin E deficient mice were much more susceptible to MPTP toxicity than controls, in terms of lethality and DOPAC depletion in the substantia nigra. However, in the same vitamin E deficient mice, the striatum was partially protected from neurotransmitter and metabolite depletion by MPTP. The mechanism of toxicity of MPTP may differ in the striatum and the midbrain.

Nicotinamide as a precursor for NAD+ prevents apoptosis in the mouse brain induced by tertiary-butylhydroperoxide.

The vitamin nicotinamide can protect against oxidative stress-induced apoptosis in the brain when used as a precursor for nicotinamide adenine dinucleotide (NAD+). The intracerebroventricular administration of tertiary-butylhydroperoxide (t-buOOH) to mice was used to simulate physiologic oxidative stress and apoptosis which may occur in some neurodegenerative conditions. t-buOOH produced characteristic apoptotic nuclear degeneration in neurons with extensive fragmentation of DNA. In this report we show that the elevation of NAD+ by nicotinamide prevents DNA fragmentation during apoptosis or necrosis in the brain as stimulated by t-buOOH administration. NAD+ levels can be increased by 50% in the brain. This may prevent the critical depletion of NAD+ by poly(ADP-ribose) polymerase (PARP) and provide additional substrate during the repair of DNA. Nicotinamide may be of particular interest in the treatment of neurodegeneration.

Apoptosis and DNA fragmentation as induced by tertiary butylhydroperoxide in the brain.

In this study, the effect of intracerebroventricular administration of the free radical generator, tertiary butylhydroperoxide, on DNA, was quantitated. Previous studies had established DNA as a very important site of free radical attack. The purpose of the study was to detect whether DNA was one of the primary targets of the toxin as well as to detect any apoptosis that may have been induced by the toxin. The DNA fragmentation assay clearly showed DNA damage within 20 min of administration of 109.7 mg/kg t-BuOOH almost in all brain regions in both 2-month and 8-month-old C57BL/6 mice. In Situ Apoptosis Detection assay, where brain sections were stained with Apoptag, demonstrated that t-BuOOH induces apoptosis in many brain regions. Electron microscopy was done to show nuclear damage and DNA fragments appearing in the cytoplasm. Cresyl violet staining was done to show that while low dose (21.9 mg/kg) t-BuOOH induces apoptosis, it may also induce necrosis in other cells of the same brain region. Thus, from this study we can conclude that DNA may be one of the primary target sites of free radical attack in the brain, and results in both necrosis and apoptosis. This can have a profound effect on neurodegeneration.

Tyrosine hydroxylase: mechanisms of oxygen radical formation.

A new mechanism of oxygen radical formation in dopaminergic neurons is proposed, based on the oxidative mechanism of tyrosine hydroxylase. The cofactor (6R,6S)-5,6,7,8-tetrahydrobiopterin can rearrange in solution which allows an autoxidation reaction producing O2.-, H2O2 and HO.. The combination of tyrosine hydroxylase and the cofactor produces more oxygen radicals than does the autoxidation of the cofactor. This production of oxygen radicals could be damaging to dopaminergic neurons. In the presence of tyrosine, the enzyme produces less radicals than it does in the absence of tyrosine. Mechanisms are proposed for the generation of reactive oxygen species during the autoxidation of the cofactor and during enzymatic catalysis. The generation, by tyrosine hydroxylase, of very small amounts of oxygen radicals over the period of 65 years could contribute to the oxidative stress that causes Parkinson's disease.

Dietary α-tocopherol content modulates responses to moderate ethanol consumption.

Rats were fed with diets containing differing amounts of α-tocopherol for 21 days. For the latter 14 days of this period, one half of the rats also received ethanol (7% v/v) in the drinking water. Treatments did not alter the rate of weight gain between groups. Hepatic glutathione levels were depressed by ethanol treatment in rats receiving diets deficient in α-tocopherol or containing normal levels of the vitamin (50 ppm). However, this depression was not found in rats maintained on a high α-tocopherol diet (1000 ppm). The high α-tocopherol diet also prevented the ethanol-induced inhibition of proteolytic activity within the liver. A dose-dependent reduction of rates of hepatic generation of reactive oxygen species was effected by this vitamin. Within the central nervous system, the only region showing an ethanol-induced lowering of glutathione levels, was the midbrain of rats receiving the α-tocopherol deficient diet.