Brain aging phenomena in migrating sockeye salmon Oncorhynchus nerka nerka.

Aging, a process occurring in all vertebrates, is closely related to a loss in physical and functional abilities. There is widespread interest in clarifying the relevance of environmental, metabolic, and genetic factors for vertebrate aging. In the Pacific salmon a dramatic example of aging is known. Looking for changes in the salmon brain, perhaps even in the role of initiating the aging processes, we investigated several biochemical parameters that should reflect brain functional activity and stress response such as the neurotransmitters dopamine, and serotonin, and two of their respective metabolites 3,4-dihydroxyphenylacetic acid, and 5-hydroxyindole acetic acid, as well as glutathione, glutathione disulfide, and the extent of terminal deoxynucleotidyltransferase-mediated dUTP nick end-labelling. The aging of migrating sockeye salmon (Oncorhynchus nerka nerka) is accompanied by gradual increase in dopamine and serotonin turnover and a gradual decrease of brain total protein and glutathione levels. There appears to be an increased need for detoxification of reactive biological intermediates since activities of superoxide dismutase and catalase increase with age. However, our data do not support a major increase in apoptotic cell death during late aging but rather implicate an age related downward regulation of protein and glutathione synthesis and proteolysis increasing the need for autophagocytosis or heterophagocytosis in the course of cell death.

(r)-, but not (s)-alpha lipoic acid stimulates deficient brain pyruvate dehydrogenase complex in vascular dementia, but not in Alzheimer dementia.

In dementia of Alzheimer type (DAT), cerebral glucose metabolism is reduced in vivo, and enzymes involved in glucose breakdown are impaired in post-mortem brain tissue. Pyruvate dehydrogenase complex activity (PDHc) is one of the enzymes known to be reduced, while succinate dehydrogenase activity (SDH), another enzyme of oxidative glucose metabolism is unchanged. In dementia of vascular type (DVT), variable changes in glucose metabolism have been demonstrated in vivo, while changes of enzyme activities in post-mortem brain tissue are unknown. Here, PDHc and SDH activity were stimulated with each of the two stereoisomers of alpha lipoic acid in post-mortem parietal brain cortex of patients with DAT, DVT, and one case of Pick's disease and compared to stimulation effects in a control group, matched for age, sex, post-mortem delay, and storage time of brain tissue. PDHc in DAT and DVT, but not in Pick's disease was reduced. PDHc activity could be slightly stimulated by 10 micro M of the physiological stereoisomer (r)-alpha-lipoic acid, in controls and DVT (possibly also in Pick's disease), but not in DAT. In all groups investigated SDH was activated by 100 micro M and 1 mM of both isomers of alpha-lipoic acid, whereas 10 mM of both stereoisomers of alpha-lipoic acid caused an inhibition of both, PDHc and SDH activity. The loss of basal and of (r)-alpha-lipoic acid stimulated PDHc activity indicate that a functional or structural impairment of PDHc may exist in DAT and DVT which is not merely attributable to loss of mitochondria since basal and stimulated SDH activities are similar in controls, DVT and DAT, thus indicating selective vulnerability of PDHc.

Altered redox state of platelet coenzyme Q10 in Parkinson's disease.

BACKGROUND: The reduced form of coenzyme Q10 (CoQ10) acts as a lipophilic antioxidant and participates in electron and proton transport of the respiratory chain in the inner mitochondrial membrane. An alteration in CoQ10 redox state may thus reflect a change in membrane electron transport and the effectiveness of defense against toxic reactive oxygen species such as hydrogen peroxide and superoxide. In Parkinson's disease alterations in the activities of complex I have been reported in substantia nigra and platelets. Deficiency of mitochondrial enzyme activities could affect electron transport which might be reflected by the platelet CoQ10 redox state. METHOD: We have determined concentrations of the reduced and oxidized forms of CoQ10 and the activity of monoamine oxidase B in platelets isolated from parkinsonian patients and age- and gender-matched controls. RESULTS: Platelet CoQ10 redox ratios (reduced CoQ10 to oxidized CoQ10) and the ratio of the reduced form, compared with total platelet CoQ10, were significantly decreased in de novo parkinsonian patients. Platelet CoQ10 redox ratios were further decreased by L-DOPA treatment (not significant), whilst selegiline treatment partially restored CoQ10 redox ratios. Monoamine oxidase activities in non-selegiline treated patients were similar to controls. INTERPRETATION: Our results either suggest an impairment of electron transport or a higher need for reduced forms of CoQ10 in the platelets of even de novo parkinsonian patients. However, the CoQ10 redox ratio was not correlated to disease severity, as determined by the Hoehn and Yahr PD disability classification, suggesting that this parameter may not be useful as a peripheral trait marker for the severity of PD but as an early state marker of PD.

Acetylcholine in human CSF: methodological considerations and levels in dementia of Alzheimer type.

Four different methods to measure acetylcholine (ACh) and choline (Ch) concentration, i.e. thermospray/mass spectroscopy (TS/MS), high pressure liquid chromatography/mass spectroscopy (HPLC/MS), high pressure liquid chromatography/electrochemical detection (HPLC/ECD) and gas chromatography/mass spectroscopy (GC/MS), both latter methods coupled to a solid phase extraction system were compared for their applicability to human lumbar cerebrospinal fluid (CSF). Furthermore, samples from 15 control persons and 11 patients with dementia of Alzheimer-type (DAT) were compared to search for an ACh deficit in the CSF in DAT. GC/MS was the most sensitive, but most laborious method, and HPLC/ECD was acceptably sensitive, reliable and more specific. TS/MS was not specific enough for CSF extracts and HPLC/MS was more specific, but far less sensitive. Thus, only GC/MS and HPLC/ECD were qualified to detect ACh in human CSF extracts. Comparison of GC/MS and HPLC/ECD revealed highly correlated levels of ACh (r = 0.999). Using HPLC/ECD, ACh concentrations were greatly reduced in the DAT group (3.75 +/- 1.40 pmol/ml CSF) as compared to the controls (6.14 +/- 1.39 pmol/ml CSF), but the difference between controls and DAT patients was not statistically significant due to the number of samples below detection limit (8 out of 11 samples in DAT, 7 out of 15 in controls). Ch concentrations were not statistically significant between the two groups. The data show that methodological limitations preclude a widespread clinical application of determining ACh in the human CSF. Despite of reductions of ACh in the CSF in DAT, the determination of Ach in the CSF is not suitable for diagnostic purposes.

Acute MPTP treatment produces no changes in mitochondrial complex activities and indices of oxidative damage in the common marmoset ex vivo one week after exposure to the toxin.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been shown to cause a Parkinsonian syndrome in man and non-human primates. Hypotheses concerning the pathogenetic mechanisms of MPTP toxicity on nigro-striatal dopaminergic neurons relate to impairment of mitochondrial function and oxidative stress. However, surprisingly few primate studies addressed these issues ex vivo. Thus, the present study assessed the enzyme activities of the respiratory chain, GSH/GSSG and ubiquinol/ubiquinone content in the MPTP primate model (common marmoset, Callithrix jacchus; 2 mg MPTP-hydrochloride/kg body wt were injected subcutaneously (s.c.) on four consecutive days; animals were sacrificed 7 days after last MPTP exposure). Activities of respiratory chain enzymes were measured in crude homogenates of the caudate nucleus, because the probable toxic metabolite of MPTP, MPP+, is transported into dopaminergic neurons via the dopamine uptake system in striatal synapses and mitochondria are concentrated in axonal terminals. Since MPP+ can damage membranes of axonal terminals of nigro-striatal neurons we measured GSH/GSSG contents in the putamen and ubiquinol/ubiquinone concentrations in the substantia nigra and putamen as indices of oxidative damage. At the time of sacrifice MPTP-induced deficits comprised severe behavioural Parkinsonian symptoms, profound depletion of striatal dopamine and its major metabolites as well as pronounced loss of nigro-striatal neurons. Despite these severe lesions, acute MPTP treatment had no effect on any of the enzymes of the respiratory chain in the caudate nucleus and indices of oxidative damage in both the substantia nigra and putamen. These results suggest that factors other than mitochondrial impairment and/or oxidative stress may be involved in MPTP neurotoxicity in primates. Alternatively, early compensatory mechanisms and/or transient effects could account for the reported results and will be discussed.

Pretreatment with nimodipine prevents MPTP-induced neurotoxicity at the nigral, but not at the striatal level in mice.

The present study assessed the effects of pretreatment with the calcium-L-type channel blocker nimodipine on biochemical and histological parameters of systemic MPTP-induced (2 x 40 mg kg-1 body weight subcutaneously, 16 h apart), dopaminergic neurotoxicity in black C57BL/6 mice. Continuous administration of nimodipine via subcutaneously implanted pellets (10 mg), starting 7 days before administration of MPTP, did not antagonize the striatal MPTP-induced dopamine depletion (caudate-putamen: 80% of control; nucleus accumbens; 25% of control), but almost completely prevented the MPTP-induced tyrosine hydroxylase immunoreactive-cell loss in the substantia nigra (20% of control) 7 days after administration of MPTP. This data suggests that pretreatment with nimodipine--during the observation period of 7 days--protects against MPTP-induced neurotoxicity in mice at the nigral ('cell body'), but not at the synaptic striatal level.

High dose selegiline augments striatal ubiquinol in mouse: an indication of decreased oxidative stress or of interference with mitochondrial respiration? A pilot study.

The effects of unspecific doses of the irreversible monoamine oxidase inhibitor selegiline on alpha-tocopherol, alpha-tocopherolquinone, ubiquinol and ubiquinone were studied in frontal cortex, hippocampus and striatum of male C57BL/6 mice 4 h and 96 h after a single or six injections of selegiline (100 mg/kg body weight, i.p.), respectively. Inhibition of monoamine oxidase was confirmed by activity measurements of its isoforms A and B in brain stem nuclei and striatum as well as by determination of striatal levels of dopamine and its major metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid. In general, levels of alpha-tocopherol were not altered and levels of alpha-tocopherolquinone were below the detection limit. However, 96 h following selegiline, levels of ubiquinols 9 and 10 were significantly increased, whereas levels of ubiquinones 9 and 10 concomitantly decreased in the striatum. Concentrations of ubiquinols and ubiquinones in frontal cortex and hippocampus were unchanged 96 h following selegiline. These data suggest that selegiline affects the striatal redox ratio of ubiquinol to ubiquinone which is important for cellular antioxidant defense and mitochondrial electron transfer.

Effect of lipoic acid on redox state of coenzyme Q in mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and diethyldithiocarbamate.

We investigated the effects of a combined treatment of male C57Bl/6 mice with diethyldithiocarbamate and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the absence or presence of different forms of lipoic acid (Thioctacid TR; commonly used for treatment of diabetic polyneuropathies) on levels and redox states of alpha-tocopherol and coenzyme Q in vivo and on activities of various enzymes of energy metabolism ex vivo. Treatment of mice with diethyldithiocarbamate plus MPTP resulted in a decrease in dopamine (67%) and its major metabolites dihydroxyphenylacetic acid (38%) and homovanillic acid (37%) in striatum. alpha-Tocopherol levels were unaltered in striatum; however, the reduced forms of coenzyme Q were decreased in frontal cortex and hippocampus following diethyldithiocarbamate plus MPTP. In frontal cortex activity of NADH dehydrogenase was significantly inhibited by diethyldithiocarbamate plus MPTP ex vivo, suggesting that the neurotoxic metabolite of MPTP, 1-methyl-4-phenylpyridinium ion, is acting in brain regions other than striatum as well. Lipoic acid, administered 6 times, each at 90 min prior to MPTP, could not restore dopamine in striatum but in contrast maintained a normal ratio of the reduced form to the oxidized form of coenzyme Q, suggesting an interaction of lipoic acid with energy metabolism which seems, however, not only to be due to an activation of pyruvate dehydrogenase.

Influence of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, lipoic acid and L-deprenyl on the interplay between cellular redox systems.

For several years there is controversy concerning the toxic potency of reaction products catalyzed by monoamine oxidase in neurodegenerative processes. There is uncertainty whether products of catecholamine oxidation are pathogenetically relevant factors for neuronal cell death in Parkinson's disease. To date products responsible for impairment of biochemical functions essential for cell viability are not yet identified, and the primary site of damage within the cell is unknown. Ammonia, aldehydes and hydrogen peroxide are formed via monoamine oxidase catalyzed oxidations of primary amines. But which of them, if any, is damaging to the cell? We discuss some aspects of the oxidative stress theory of cell degeneration in relation to toxicity of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and to monoamine oxidation. Furthermore, we consider possible functional relationships of mitochondrial electron transfer reactions, toxicity of MPTP and MAO activity.

Effects of long-term recovery from transient cerebral ischemia in rat brain: tissue levels of acetylcholine, monoamines, and their metabolites.

Concentrations of acetylcholine and the monoaminergic neurotransmitters dopamine, serotonin and their respective metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), 4-hydroxy-3-methoxy-phenylacetic acid (HVA), 5-hydroxyindolacetic acid (5-HIAA) and choline were simultaneously determined in the corpus striatum of rats after 15 min. complete cerebral ischemia (CCI) and in different intervals (1, 24, 48, 72, 96 hours) of postischemic cerebral reperfusion. Results were compared to respective sham-operated control animals. After 15 min. CCI acetylcholine concentration decreased to 15%, and dopamine concentration to 56% of the control values. The metabolite levels of DOPAC decreased to 40% and HVA to 64% of the control values. Acetylcholine, dopamine, serotonin and choline concentrations were not changed significantly after reperfusion. The metabolites HVA and 5-HIAA showed their maximum increases after 1 and 24 hours of reperfusion, additionally HVA was decreased both, after 72 and 96 hours of reperfusion. The data indicate that surprisingly little permanent damage could be caused by a 15 min. ischemia in the striatum. Tissue levels of the neurotransmitters appeared differentially altered but similarly regulated during ischemia and subsequent recirculation. Acetylcholine and dopamine levels decreased profoundly during ischemia. However, acetylcholine levels could be compensated rapidly during reperfusion, whereas the dopaminergic system showed a long-lasting change in its turnover rate. Although serotonin levels were unaffected by CCI, there was an increase of its presumed turnover rate during reperfusion.

The thiobarbituric acid assay reflects susceptibility to oxygen induced lipid peroxidation in vitro rather than levels of lipid hydroperoxides in vivo: a methodological approach.

Although the hypothesis of oxidative stress as a pathogenetic factor of neurodegenerative diseases became a matter of interest recently, direct evidence supporting this hypothesis is rare. The most prominent assay being currently used as an index for lipid peroxidation products in vivo is the thiobarbituric acid assay. Thiobarbituric acid reactive substances are mainly formed during the decomposition of lipid hydroperoxides in vitro. It is questionable however, that all species detectable with thiobarbituric acid are derived from in vivo preformed lipid hydroperoxides. These studies were undertaken to investigate the influence of autoxidation reactions on colour production during the acid heating stage of the assay. If driven aerobically, more than 90% of thiobarbituric acid reactive substances are newly generated in vitro during incubation at 95 degrees C for 75 min. This process can be enhanced by addition of ferric iron. Chain breaking antioxidants like butylated hydroxytoluene decrease colour formation in the absence or in the presence of iron. If driven anaerobically under argon, colour formation was only 10% of aerobically heated homogenates or lipid extracts of human brain tissue. These results may indicate that measurement of thiobarbituric acid reactive substances under the aerobic conditions described here reflects to a great extent the susceptibility of brain tissue or lipids to oxygen-induced formation of lipid hydroperoxides in vitro rather than degradation products of in vivo performed lipid hydroperoxides.

Mutagenic nitrenes/nitrenium ions from azido-imidazoarenes and their structure-activity relationships.

New heterocyclic arylazides (azido-imidazoarenes) structurally related to the food mutagen/carcinogen 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) have been prepared. Their photolysis yields nitrenes and/or nitrenium ions which induce mutations in Salmonella typhimurium. The relationships between the chemical structure and mutagenic activity of these species are the same as those found in our previous studies of the amino- and nitro-imidazoarenes. Therefore the efficiency of the reaction with DNA of the ultimate metabolite, the nitrene/nitrenium ion, is the critical step governing the mutagenic potency of the amino- and nitro-imidazoarenes. The efficiency of DNA-binding depends on the delocalization of the positive charge of the nitrenium ion or of the electron deficiency of the nitrene. It is typical of the N-1-substituted and N-3-substituted arenimidazolyl-nitrenium ions that they can form another nitrenium resonance structure very similar to the parent nitrenium ion structure. We suggest that this property of the nitrene/nitrenium ion, in combination with its aromatic structure facilitating carbonium ion resonance structures, is the basic reason for the extremely potent mutagenic activity of IQ and related food mutagens/carcinogens.

Photolysis of arylazides and generation of highly electrophilic DNA-binding and mutagenic intermediates.

Photolysis of arylazides with long wavelength ultraviolet (NUV) light in an aqueous medium produces short-lived reactive chemical species which bind to DNA and deoxynucleoside 3'-phosphates and induce reversion mutations in frameshift tester strains of Salmonella typhimurium. Nitrenes are known reactive products of azide photolysis, so the DNA-binding and mutagenic species is either a nitrene or a nitrene-derivative. An N-hydroxyarylamine intermediate, potentially formed from a nitrene and water, can be excluded because the mutagenic potencies of the reactive species in TA98 and in the hydroxylamine-resistant TA98/1,8-DNP6 are of the same order, and because the life-time of this species is very short. The mutagenic potency of the arylazide photolysis products decreases in the order azido-IQ greater than 1-azidopyrene greater than azido-MeIQx greater than 6-azidochrysene greater than 2-azidofluorene greater than 4-azidobiphenyl greater than 2-azido-naphthalene greater than 1-azido-naphthalene. This potency sequence correlates with that of the corresponding arylamines. Furthermore, their DNA binding products are chromatographically identical with those obtained in cellular, metabolizing systems from nitroarenes and arylamines. Therefore, the reactive, electrophilic azide photolysis product is very likely a nitrenium ion formed by protonation of a nitrene. Nitrenium ions are also the ultimate mutagens/carcinogens formed from nitroarenes and arylamines. Arylazides can therefore be considered as stabilized forms of arylnitrenium ions. The arylazide-nitrene technique reported here is new and simple and provides ready access to presumed nitrenium ions which are otherwise difficult to obtain.

Synthesis of 2-azido-3-methylimidazo[4,5-f]quinoline and photolytic generation of a highly reactive and mutagenic IQ derivative.

Azido-IQ (2-azido-3-methylimidazo[4,5-f]quinoline), a novel analog of the food mutagen and carcinogen IQ (2-amino-3-methylimidazo[4,5-f]quinoline) was synthesized and characterized. Both thermolysis and photolysis of this azide yield a short-lived nitrene which can react as an electrophile either directly or via protonation to a nitrenium ion. Reaction of the nitrene/nitrenium ion with water produces N-hydroxy-IQ; reactions with nucleotides and with DNA (in vitro and in cells) produce adducts efficiently. Correspondingly, high frequencies of mutations are induced in Salmonella typhimurium by photolyzed azido-IQ. Comparative mutation assays with the S. typhimurium strains TA98 and the hydroxylamine-resistant TA98/1,8-DNP6 provide evidence for a novel mechanism of mutation, direct reaction of the nitrene or nitrene-derived nitrenium ion with DNA without involvement of the hydroxylamine. The photolysis of arylazides promises to be a very convenient and generally applicable non-enzymatic procedure for the cell-free or intracellular generation of short-lived and highly reactive electrophilic species which are assumed to be identical with the metabolically formed ultimate mutagens/carcinogens of arylamines and nitroarenes.

Synthesis and mutagenic activity of nitro-imidazoarenes. A study on the mechanism of the genotoxicity of heterocyclic arylamines and nitroarenes.

A series of nitro-imidazoarenes (nitro-IAs) were synthesized from the corresponding amino-imidazoarenes (amino-IAs). These two classes of compounds are structurally related to the potent food mutagen and carcinogen, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). The mutagenic activities of the nitro-IAs were assayed in the Salmonella typhimurium frameshift tester strains TA98, TA98/1,8-DNP6 and TA98NR without use of extracellular metabolization. Nitro-IQ, the nitro counterpart of IQ, was two times more mutagenic than IQ. In general, the mutagenic activities of the nitro-IAs varied over 50,000-fold. The relationships between the chemical structures and mutagenic activities are identical with those previously reported for the corresponding amino-IAs: the methyl group on the imidazole ring and the quinoline-nitrogen were found to be required for potent mutagenic activity. The reductive activation of the nitro-IAs is not carried out primarily by the 'classical' nitroreductase of Salmonella which is defective in TA98NR. The O-acetyltransferase defective in TA98/1,8-DNP6 is required for the efficient production of the ultimate mutagens of the nitro-IAs. The interchangeability of the structure-activity relationships of the nitro-IAs and amino-IAs reflects a basic similarity of the mechanisms of the mutagenicity of the two classes of compounds. It is likely that N-hydroxy compounds are proximate metabolites common to the nitro-IAs and amino-IAs; they are further activated by an acetyl-CoA-dependent O-acetyltransferase of Salmonella. It is very likely a property of the ultimate mutagen, possibly a nitrenium ion, which governs the mutagenic potency of the different nitro- and amino-IAs and thus determines the structure-activity relationships.