Protective effect of harmalol and harmaline on MPTP neurotoxicity in the mouse and dopamine-induced damage of brain mitochondria and PC12 cells

The present study elucidated the protective effect of beta-carbolines (harmaline, harmalol, and harmine) on oxidative neuronal damage. MPTP treatment increased activities of total superoxide dismutase, catalase, and glutathione peroxidase and levels of malondialdehyde and carbonyls in the basal ganglia, diencephalon plus midbrain of brain compared with control mouse brain. Coadministration of harmalol (48 mg/kg) attenuated the MPTP effect on the enzyme activities and formation of tissue peroxidation products. Harmaline, harmalol, and harmine attenuated both the 500 microM MPP(+)-induced inhibition of electron flow and membrane potential formation and the 100 microM dopamine-induced thiol oxidation and carbonyl formation in mitochondria. The scavenging action of beta-carbolines on hydroxyl radicals was represented by inhibition of 2-deoxy-D-ribose degradation. Harmaline and harmalol (100 microM) attenuated 200 microM dopamine-induced viability loss in PC12 cells. The beta-carbolines (50 microM) attenuated 50 microM dopamine-induced apoptosis in PC12 cells. The compounds alone did not exhibit significant cytotoxic effects. The results indicate that beta-carbolines attenuate brain damage in mice treated with MPTP and MPP(+)-induced mitochondrial damage. The compounds may prevent dopamine-induced mitochondrial damage and PC12 cell death through a scavenging action on reactive oxygen species and inhibition of monoamine oxidase and thiol oxidation.     

Differential effect of catecholamines and MPP(+) on membrane permeability in brain mitochondria and cell viability in PC12 cells.

The present study examined the effect of dopamine, 6-hydroxydopamine (6-OHDA), and MPP(+) on the membrane permeability transition in brain mitochondria and on viability in PC12 cells. Dopamine and 6-hydroxydopamine induced the swelling and membrane potential change in mitochondria, which was inhibited by addition of antioxidant enzymes, SOD and catalase. In contrast, antioxidant enzymes did not reduce the effect of MPP(+) on mitochondrial swelling and membrane potential. Catecholamines enhanced the Ca(2+) uptake and release by mitochondria, and the addition of MPP(+) induced Ca(2+) release. Catecholamines induced a thiol oxidation in mitochondria that was decreased by antioxidant enzymes. MPP(+) showed a little effect on the cytochrome c release from mitochondria and did not induce thiol oxidation. Catecholamines and MPP(+) induced a cell death, including apoptosis, in PC12 cells that was inhibited by addition of antioxidant enzymes. The result suggests that the oxidation of dopamine and 6-hydroxydopamine could modulate the membrane permeability in brain mitochondria and induce PC12 cell death, which may be ascribed to oxidative stress. MPP(+) appears to exert a toxic effect on neuronal cells by the action, which is different from catecholamines.     

Combined effect of dopamine and MPP+ on membrane permeability in mitochondria and cell viability in PC12 cells

The present study examined the combined effect of dopamine and 1-methyl-4-phenylpyridinium (MPP(+)) on the membrane permeability in isolated brain mitochondria and on cell viability in PC12 cells. MPP(+) increased effect of dopamine against the swelling, membrane potential, and Ca(2+) transport in isolated mitochondria, which was not inhibited by the addition of antioxidant enzymes (SOD and catalase). Dopamine or MPP(+) caused the decrease in transmembrane potential, increase in reactive oxygen species, depletion of GSH, and cell death in PC12 cells. Antioxidant enzymes reduced each effect of dopamine and MPP(+) against PC12 cells. Co-addition of dopamine and MPP(+) caused the decrease in the transmembrane potential and increase in the formation of reactive oxygen species in PC12 cells, in which they showed an additive effect. Dopamine plus MPP(+)-induced the depletion of GSH and cell death in PC12 cells were not decreased by the addition of antioxidant enzymes, rutin, diethylstilbestrol, and ascorbate. Melanin caused a cell viability loss in PC12 cells. The N-acetylcysteine, N-phenylthiourea, and 5-hydroxyindole decreased the cell death and the formation of dopamine quinone and melanin induced by co-addition of dopamine and MPP(+), whereas deprenyl and chlorgyline did not show an inhibitory effect. The results suggest that co-addition of dopamine and MPP(+) shows an enhancing effect on the change in mitochondrial membrane permeability and cell death, which may be accomplished by toxic quinone and melanin derived from the MPP(+)-stimulated dopamine oxidation.     

Modification of Dopamine Transporter Function: Effect of Reactive Oxygen Species and Dopamine

Abstract: Dopamine can oxidize to form reactive oxygen species and quinones, and we have previously shown that dopamine quinones bind covalently to cysteinyl residues on striatal proteins. The dopamine transporter is one of the proteins at risk for this modification, because it has a high affinity for dopamine and contains several cysteinyl residues. Therefore, we tested whether dopamine transport in rat striatal synaptosomes could be affected by generators of reactive oxygen species, including dopamine. Uptake of [³H]dopamine (250 n M ) was inhibited by ascorbate (0.85 m M ; −44%), and this inhibition was prevented by the iron chelator diethylenetriaminepentaacetic acid (1 m M ), suggesting that ascorbate was acting as a prooxidant in the presence of iron. Preincubation with xanthine (500 µ M ) and xanthine oxidase (50 mU/ml) also reduced [³H]dopamine uptake (−76%). Preincubation with dopamine (100 µ M ) caused a 60% inhibition of subsequent [³H]dopamine uptake. This dopamine-induced inhibition was attenuated by diethylenetriaminepentaacetic acid (1 m M ), which can prevent iron-catalyzed oxidation of dopamine during the preincubation, but was unaffected by the monoamine oxidase inhibitor pargyline (10 µ M ). None of these incubations caused a loss of membrane integrity as indicated by lactate dehydrogenase release. These findings suggest that reactive oxygen species and possibly dopamine quinones can modify dopamine transport function.     

Interaction between 6-hydroxydopamine and transferrin: "Let my iron go"

The dopamine analogue 6-hydroxydopamine (6-OHDA) is selectively toxic to catecholaminergic neurons. Because of its selectivity for neuroblastic cells in the sympathetic nervous system lineage, 6-OHDA has been suggested as a chemotherapeutic agent for targeted treatment of patients with neuroblastoma. We tested the hypothesis that the toxicity of 6-OHDA is caused by its interaction with serum ferric transferrin (Fe-TF) resulting in release of iron. We further hypothesized that this iron, through its redox-cycling by 6-OHDA, triggers generation of reactive oxygen species. 6-OHDA-induced release of iron from Fe-TF was demonstrated by: (1) low-temperature EPR spectroscopic evidence for decay of the characteristic Fe-TF signal (g = 4.3) and appearance of the high-spin signal from iron chelated by 6-OHDA oxidation products; (2) spectrophotometric detection of complexing of iron with the Fe(2+) chelator ferrozine; (3) redox-cycling of ascorbate yielding EPR-detectable ascorbate radicals; and (4) generation of hydroxyl radicals as evidenced by EPR spectroscopy of their adduct with a spin trap, 5, 5'-dimethylpyrroline oxide (DMPO) (DMPO-OH). Our low-temperature EPR studies showed that in human plasma, 6-OHDA caused iron release only under nitrogen gas but not under air or oxygen. The absence of a 6-OHDA effect in plasma under aerobic conditions was most likely due to its ferroxidase activity [with consequent reuptake of Fe(III) by apoTF] and catalytic oxidation of 6-OHDA by ceruloplasmin. Modeling of these plasma activities by a stable nitroxide radical, 2,2,6, 6-tetramethyl-1-piperidinyloxy (TEMPOL), resulted in protection of plasma Fe-TF against iron release under nitrogen. Parenteral administration of 6-OHDA to mice resulted in iron release from Fe-TF as evidenced by transformation of the Fe-TF low-temperature EPR signal that was indistinguishable from that seen in in vitro models. In addition, administration of the iron chelator deferoxamine (DFO) to mice prior to administration of toxic doses of 6-OHDA resulted in a decrease in activity impairment of mice as compared to that seen with 6-OHDA alone. These findings underscore the physiological and pharmacological relevance of 6-OHDA-mediated iron release from Fe-TF and suggest that iron chelators (DFO) may be used for prevention of 6-OHDA toxicity.     

Mitochondrial dysfunction mediated by quinone oxidation products of dopamine: Implications in dopamine cytotoxicity and pathogenesis of Parkinson's disease

The study has demonstrated that dopamine induces membrane depolarization and a loss of phosphorylation capacity in dose-dependent manner in isolated rat brain mitochondria during extended in vitro incubation and the phenomena are not prevented by oxyradical scavengers or metal chelators. Dopamine effects on brain mitochondria are, however, markedly prevented by reduced glutathione and N-acetyl cysteine and promoted by tyrosinase present in the incubation medium. The results imply that quinone oxidation products of dopamine are involved in mitochondrial damage under this condition. When PC12 cells are exposed to dopamine in varying concentrations (100-400μM) for up to 24h, a pronounced impairment of mitochondrial bio-energetic functions at several levels is observed along with a significant (nearly 40%) loss of cell viability with features of apoptotic nuclear changes and increased activities of caspase 3 and caspase 9 and all these effects of dopamine are remarkably prevented by N-acetyl cysteine. N-acetyl cysteine also blocks nearly completely the dopamine induced increase in reactive oxygen species production and the formation of quinoprotein adducts in mitochondrial fraction within PC12 cells and also the accumulation of quinone products in the culture medium. Clorgyline, an inhibitor of MAO-A, markedly decreases the formation of reactive oxygen species in PC12 cells upon dopamine exposure but has only mild protective actions against quinoprotein adduct formation, mitochondrial dysfunctions, cell death and caspase activation induced by dopamine. The results have indicated that quinone oxidation products and not reactive oxygen species are primarily involved in cytotoxic effects of dopamine and the mitochondrial impairment plays a central role in the latter process. The data have clear implications in the pathogenesis of Parkinson's disease.     

Photocatalytic actions of the pesticide metabolite 2-hydroxyquinoxaline: destruction of antioxidant vitamins and biogenic amines - implications of organic redox cycling

Toxicity of the pesticide quinalphos may comprise secondary, delayed effects by its main metabolite 2-hydroxyquinoxaline (HQO). We demonstrate that HQO can destroy photocatalytically vitamins C and E, catecholamines, serotonin, melatonin, the melatonin metabolite AMK (N(1)-acetyl-5-methoxykynuramine), and unsubstituted and substituted anthranilic acids when exposed to visible light. In order to avoid HQO-independent ascorbate oxidation by light and to exclude actions by hydroxyl radicals, experiments on this vitamin were carried out in ethanolic solutions. Other substances tested (vitamin E, melatonin, anthranilic acids) were also photocatalytically destroyed by HQO in ethanol. After product analyses had indicated that HQO was not, or only poorly, degraded in the light, despite its catalytic action on other compounds, we followed directly the time course of HQO and ascorbate concentrations in ethanol. While ascorbate was largely destroyed, no change in HQO was demonstrable within 2 h of incubation. Destruction was not prevented by the singlet oxygen quencher DABCO. Obviously, HQO is capable of undergoing a process of organic redox cycling, perhaps via an intermediate quinoxaline-2-oxyl radical. Health problems from HQO intoxication may not only arise from the loss of valuable biomolecules, such as antioxidant vitamins and biogenic amines, but also from the formation of potentially toxic products. Dimerization and oligomerization are involved in several oxidation processes catalyzed by HQO, especially in the indoleamines, in dopamine, and presumably also in vitamin E. Melatonin oxidation by HQO did not only lead to the well-known - and usually protective - metabolite AFMK (N(1)-acetyl-N(2)-formyl-5-methoxykynuramine), but also to a high number of additional products, among them dimers and trimers. DABCO did not prevent melatonin destruction, but changed the spectrum of products. Serotonin was preferentially converted to a dimer, which can further oligomerize. Several indole dimers are known to be highly neurotoxic, as well as oxidation products formed from catecholamines via the adrenochrome/noradrenochrome pathway. Destruction of melatonin may cause deficiencies in circadian physiology, in immune functions and in antioxidative protection.     

Mitochondrial dysfunction mediated by quinone oxidation products of dopamine: Implications in dopamine cytotoxicity and pathogenesis of Parkinson's disease

The study has demonstrated that dopamine induces membrane depolarization and a loss of phosphorylation capacity in dose-dependent manner in isolated rat brain mitochondria during extended in vitro incubation and the phenomena are not prevented by oxyradical scavengers or metal chelators. Dopamine effects on brain mitochondria are, however, markedly prevented by reduced glutathione and N-acetyl cysteine and promoted by tyrosinase present in the incubation medium. The results imply that quinone oxidation products of dopamine are involved in mitochondrial damage under this condition. When PC12 cells are exposed to dopamine in varying concentrations (100-400 μM) for up to 24 h, a pronounced impairment of mitochondrial bio-energetic functions at several levels is observed along with a significant (nearly 40%) loss of cell viability with features of apoptotic nuclear changes and increased activities of caspase 3 and caspase 9 and all these effects of dopamine are remarkably prevented by N-acetyl cysteine. N-acetyl cysteine also blocks nearly completely the dopamine induced increase in reactive oxygen species production and the formation of quinoprotein adducts in mitochondrial fraction within PC12 cells and also the accumulation of quinone products in the culture medium. Clorgyline, an inhibitor of MAO-A, markedly decreases the formation of reactive oxygen species in PC12 cells upon dopamine exposure but has only mild protective actions against quinoprotein adduct formation, mitochondrial dysfunctions, cell death and caspase activation induced by dopamine. The results have indicated that quinone oxidation products and not reactive oxygen species are primarily involved in cytotoxic effects of dopamine and the mitochondrial impairment plays a central role in the latter process. The data have clear implications in the pathogenesis of Parkinson's disease.     

Protective Effect of 1-Methylated β-Carbolines Against 3-Morpholinosydnonimine-Induced Mitochondrial Damage and Cell Viability Loss in PC12 Cells

The present study investigated the effect of 1-methylated beta-carbolines (harmaline, harmalol and harmine) on change in the mitochondrial membrane permeability and cell death due to reactive nitrogen species in differentiated PC12 cells. beta-Carbolines, caspase inhibitors (z-LEHD.fmk and z-DQMD.fmk) and antioxidants (N-acetylcysteine, dithiothreitol, melatonin, carboxy-PTIO and uric acid) depressed cell viability loss due to 3-morpholinosydnonimine (SIN-1) in PC12 cells. beta-Carbolines inhibited the nuclear damage, the decrease in mitochondrial transmembrane potential, the cytochrome c release, the formation of reactive oxygen species and the depletion of GSH caused by SIN-1 in PC12 cells. beta-Carbolines decreased the SIN-1-induced formations of 3-nitrotyrosine, malondialdehyde and carbonyls in PC12 cells. The results show that 1-methylated beta-carbolines attenuate SIN-1-induced mitochondrial damage. This results in the inhibition of caspase-9 and -3 and apoptotic cell death in PC12 cells by suppressing the toxic actions of reactive oxygen and nitrogen species, including the GSH depletion.     

Copper induced oxidation of serotonin: analysis of products and toxicity

Serotonin is a major neurotransmitter that controls many functions, ranging from mood and behaviour through to sleep and motor functions. The non-enzymatic oxidation of serotonin is of significant importance as some oxidation products are considered to be neurotoxic. An interaction between copper and serotonin has been suggested by symptoms observed in a number of neurodegenerative diseases such as Wilson's and Prion diseases. Using PC12 cells as a model of neuronal cells, we show that the interaction between copper and serotonin is toxic to undifferentiated cells. The toxicity is largely due to reactive oxygen species as cell death is significantly reduced in the presence of the antioxidant mannitol. Differentiation of the PC12 cells also confers resistance to the oxidative process. In vitro oxidation of serotonin by copper results in the eventual formation of a coloured pigment, thought to be a melanin-like polymeric species. Using spectroscopic methods we provide evidence for the formation of a single intermediate product. This dimeric intermediate was identified and characterized as 5,5'-dihydroxy-4,4'-bitryptamine. These results indicate that copper structurally alters serotonin and this process may play a role in copper related neurodegenerative diseases.     

Role of vitamin E in ascorbate-dependent protein thiol oxidation in rat liver endoplasmic reticulum

Addition of ascorbate or its generation from gulonolactone causes the oxidation of protein thiols and a simultaneous dehydroascorbate formation in rat liver microsomes. The participation of vitamin E in the phenomenon was studied. We measured ascorbate and protein thiol oxidation and lipid peroxidation in vitamin E deficient liver microsomes. Vitamin E deficiency partly uncoupled the two processes: ascorbate oxidation increased, while protein thiol oxidation decreased. These changes were accompanied with an accelerated lipid peroxidation in the vitamin E-deficient microsomes, which indicates the accumulation of reactive oxygen species. All these effects were reduced by the in vitro addition of vitamin E to the deficient microsomes, supporting its direct role in the process. The results demonstrate that vitamin E is a component of the protein thiol oxidizing machinery in the hepatic endoplasmic reticulum transferring electrons from the thiol groups towards oxygen.     

Antioxidant role of amyloid β protein in cell-free and biological systems: implication for the pathogenesis of Alzheimerdisease

In contrast to many studies showing the pro-oxidative nature of amyloid peptide, this work shows that aggregated Aβ42 peptide in varying concentrations (2–20 μM) in cell-free systems inhibits the formation of hydroxyl radicals and H₂O₂ from a mixture of iron (20 μM FeSO₄) and ascorbate (2 mM) as measured by benzoate hydroxylation assay and coumarin carboxylic acid assay. Aggregated Aβ42 in similar concentrations further prevents protein and lipid oxidation in isolated rat brain mitochondria incubated alone or with FeSO₄ and ascorbate. Moreover, mitochondria exposed to FeSO₄ and ascorbate show enhanced formation of reactive oxygen species and this phenomenon is also abolished by aggregated Aβ42. It is suggested that the antioxidant property of Aβ42 in various systems is mediated by metal chelation and it is nearly as potent as a typical metal chelator, such as diethylenetriaminepentaacetic acid, in preventing oxidative damage. However, aggregated Aβ42 causes mitochondrial functional impairment in the form of membrane depolarization and a loss of phosphorylation capacity without involving reactive oxygen species in the process. Thus, the present results suggest that the amyloid peptide exhibits a protective antioxidant role in biological systems, but also has toxic actions independent of oxidative stress.     

Human albumin prevents 6-hydroxydopamine-induced loss of tyrosine hydroxylase in in vitro and in vivo

Human albumin has recently been demonstrated to protect brain neurons from injury in rat ischemic brain. However, there is no information available about whether human albumin can prevent loss of tyrosine hydroxylase (TH) expression of dopaminergic (DA) neurons induced by 6-hydroxydopamine (6-OHDA) toxicity that is most commonly used to create a rat model of Parkinson's disease (PD). In the present study, two microliters of 1.25% human albumin were stereotaxically injected into the right striatum of rats one day before or 7 days after the 6-OHDA lesion in the same side. D-Amphetamine-induced rotational asymmetry was measured 7 days, 3 and 10 weeks after 6-OHDA lesion. We observed that intrastriatal administration of human albumin significantly reduced the degree of rotational asymmetry. The number of TH-immunoreactive neurons present in the substantia nigra was greater in 6-OHDA lesioned rats following human albumin-treatment than non-human albumin treatment. TH-immunoreactivity in the 6-OHDA-lesioned striatum was also significantly increased in the human albumin-treated rats. To examine the mechanisms underlying the effects of human albumin, we challenged PC12 cells with 6-OHDA as an in vitro model of PD. Incubation with human albumin prevented 6-OHDA-induced reduction of cell viability in PC12 cell cultures, as measured by MTT assay. Furthermore, human albumin reduced 6-OHDA-induced formation of reactive oxygen species (ROS) and apoptosis in cultured PC12 cells, as assessed by flow cytometry. Western blot analysis showed that human albumin inhibited 6-OHDA-induced activation of JNK, c-Jun, ERK, and p38 mitogen-activated protein kinases (MAPK) signaling in PC12 cultures challenged with 6-OHDA. Human albumin may protect against 6-OHDA toxicity by influencing MAPK pathway followed by anti-ROS formation and anti-apoptosis.     

Mechanisms underlying iron and copper ions toxicity in biological systems: Pro-oxidant activity and protein-binding effects

Iron and copper ions, in their unbound form, may lead to the generation of reactive oxygen species via Haber–Weiss and/or Fenton reactions. In addition, it has been shown that copper ions can irreversibly and non-specifically bind to thiol groups in proteins. This non-specific binding property has not been fully addressed for iron ions. Thus, the present study compares both the pro-oxidant and the non-specific binding properties of Fe³⁺ and Cu²⁺, using rat liver cytosol and microsomes as biological systems. Our data show that, in the absence of proteins, Cu²⁺/ascorbate elicited more oxygen consumption than Fe³⁺/ascorbate under identical conditions. Presence of cytosolic and microsomal protein, however, differentially altered oxygen consumption patterns. In addition, Cu²⁺/ascorbate increased microsomal lipid peroxidation and decreased cytosolic and microsomal content of thiol groups more efficiently than Fe³⁺/ascorbate. Finally, Fe³⁺/ascorbate and Cu²⁺/ascorbate inhibited in different ways cytosolic and microsomal glutathione S-transferase (GST) activities, which are differentially sensitive to oxidants. Moreover, in the absence of ascorbate, only Cu²⁺ decreased the content of cytosolic and microsomal thiol groups and inhibited cytosolic and microsomal GST activities. Catechin partially prevented the damage to thiol groups elicited by Fe³⁺/ascorbate and Cu²⁺/ascorbate but not by Cu²⁺ alone. N-Acetylcysteine completely prevented the damage elicited by Cu²⁺/ascorbate, Fe³⁺/ascorbate and Cu²⁺ alone. N-Acetylcysteine also completely reversed the damage to thiol groups elicited by Fe³⁺/ascorbate, partially reversed that of Cu²⁺/ascorbate but failed to reverse the damage promoted by Cu²⁺ alone. Our data are discussed in terms to the potential damage that the accumulation of iron and copper ions can promote in biological systems.     

Mitochondrial function is differentially affected upon oxidative stress

The mechanisms that lead to mitochondrial damage under oxidative stress conditions were examined in synaptosomes treated with ascorbate/iron. A loss of membrane integrity, evaluated by electron microscopy and by LDH leakage, was observed in peroxidized synaptosomes and it was prevented by pre-incubation with vitamin E (150 μM) and idebenone (50 μM). ATP levels decreased, in synaptosomes exposed to ascorbate/iron, as compared to controls. NADH-ubiquinone oxidoreductase (Cx I) and cytochrome c oxidase (Cx IV) activities were unchanged after ascorbate/iron treatment, whereas succinate-ubiquinone oxidoreductase (Cx II), ubiquinol cytochrome c reductase (Cx III) and ATP-synthase (Cx V) activities were reduced by 55%, 40%, and 55%, respectively. The decrease of complex II and ATP-synthase activities was prevented by reduced glutathione (GSH), whereas the other antioxidants tested (vitamin E and idebenone) were ineffective. However, vitamin E, idebenone and GSH prevented the reduction of complex III activity observed in synaptosomes treated with ascorbate/iron. GSH protective effect suggests that the oxidation of protein SH-groups is involved in the inhibition of complexes II, III and V activity, whereas vitamin E and idebenone protection suggests that membrane lipid peroxidation is also involved in the reduction of complex III activity. These results may indicate that the inhibition of the mitochondrial respiratory chain enzymatic complexes, that are differentially affected by oxidative stress, can be recovered by specific antioxidants.     

Protein and thiol oxidation in cells exposed to peroxyl radicals is inhibited by the macrophage synthesised pterin 7,8-dihydroneopterin

Monocyte cells are exposed to a range of reactive oxygen species (ROS) when they are recruited to a site of inflammation. In this study, we have examined the damage caused to the monocyte-like cell line U937 by peroxyl radicals and characterised the protective effect of the macrophage synthesised compound 7,8-dihydroneopterin.Exposure of U937 cells to peroxyl radicals, generated by the thermolytic breakdown of 2,2'-azobis(amidinopropane) dihydrochloride (AAPH), resulted in the loss of cell viability as measured by thiazolyl blue (MTT) reduction, and lactate dehydrogenase (LDH) leakage. The major form of cellular damage observed was cellular thiol loss and the formation of reactive protein hydroperoxides. Peroxyl radical oxidation of the cells only caused a small increase in cellular lipid oxidation measured. Supplementation of the media with increasing concentrations of 7,8-dihydroneopterin significantly reduced the cellular thiol loss and inhibited the formation of the protein hydroperoxides. High performance liquid chromatography (HPLC) analysis showed 7,8-dihydroneopterin was oxidised by both peroxyl radicals and preformed protein hydroperoxides to predominately 7,8-dihydroxanthopterin.The possibility that 7,8-dihydroneopterin is a cellular antioxidant protecting macrophage proteins during inflammation is discussed.     

Photocatalytic actions of the pesticide metabolite 2-hydroxyquinoxaline: destruction of antioxidant vitamins and biogenic amines – implications of organic redox cycling

Abstract

Toxicity of the pesticide quinalphos may comprise secondary, delayed effects by its main metabolite 2-hydroxyquinoxaline (HQO). We demonstrate that HQO can destroy photocatalytically vitamins C and E, catecholamines, serotonin, melatonin, the melatonin metabolite AMK (N¹-acetyl-5-methoxykynuramine), and unsubstituted and substituted anthranilic acids when exposed to visible light. In order to avoid HQO-independent ascorbate oxidation by light and to exclude actions by hydroxyl radicals, experiments on this vitamin were carried out in ethanolic solutions. Other substances tested (vitamin E, melatonin, anthranilic acids) were also photocatalytically destroyed by HQO in ethanol. After product analyses had indicated that HQO was not, or only poorly, degraded in the light, despite its catalytic action on other compounds, we followed directly the time course of HQO and ascorbate concentrations in ethanol. While ascorbate was largely destroyed, no change in HQO was demonstrable within 2 h of incubation. Destruction was not prevented by the singlet oxygen quencher DABCO. Obviously, HQO is capable of undergoing a process of organic redox cycling, perhaps via an intermediate quinoxaline-2-oxyl radical. Health problems from HQO intoxication may not only arise from the loss of valuable biomolecules, such as antioxidant vitamins and biogenic amines, but also from the formation of potentially toxic products. Dimerization and oligomerization are involved in several oxidation processes catalyzed by HQO, especially in the indoleamines, in dopamine, and presumably also in vitamin E. Melatonin oxidation by HQO did not only lead to the well-known – and usually protective – metabolite AFMK (N¹-acetyl-N²-formyl-5-methoxykynuramine), but also to a high number of additional products, among them dimers and trimers. DABCO did not prevent melatonin destruction, but changed the spectrum of products. Serotonin was preferentially converted to a dimer, which can further oligomerize. Several indole dimers are known to be highly neurotoxic, as well as oxidation products formed from catecholamines via the adrenochrome/noradrenochrome pathway. Destruction of melatonin may cause deficiencies in circadian physiology, in immune functions and in antioxidative protection.     

Effect of iron and manganese on hydroxyl radical production by 6-hydroxydopamine: mediation of antioxidants

6-Hydroxydopamine (6-OHDA) neurotoxicity has often been related to the generation of free radicals. Here we examined the effect of the presence of iron (Fe(2+) and Fe(3+)) and manganese and the mediation of ascorbate, L-cysteine (CySH), glutathione (GSH), and N-acetyl-CySH on hydroxyl radical (*OH) production during 6-OHDA autoxidation. In vitro, the presence of 800 nM iron increased (> 100%) the production of *OH by 5 microM 6-OHDA while Mn(2+) caused a significant reduction (72%). The presence of ascorbate (100 microM) induced a continuous generation of *OH while the presence of sulfhydryl reductants (100 microM) limited this production to the first minutes of the reaction. In general, the combined action of metal + antioxidant increased the *OH production, this effect being particularly significant (> 400%) with iron + ascorbate. In vivo, tyrosine hydroxylase immunohistochemistry revealed that intrastriatal injections of rats with 6-OHDA (30 nmol) + ascorbate (600 nmol), 6-OHDA + ascorbate + Fe(2+) (5 nmol), and 6-OHDA + ascorbate + Mn(2+) (5 nmol) caused large striatal lesions, which were markedly reduced (60%) by the substitution of ascorbate by CySH. Injections of Fe(2+) or Mn(2+) alone showed no significant difference to those of saline. These results clearly demonstrate the role of ascorbate as an essential element for the neurotoxicity of 6-OHDA, as well as the diminishing action of sulfhydryl reductants, and the negligible effect of iron and manganese on 6-OHDA neurotoxicity.     

黄连素预处理对6-羟基多巴胺所致PC12细胞损伤的影响

目的:观察黄连素(Berberine,BBR)预处理对6-羟基多巴胺(6-hydroxydopamine,6-OHDA)诱导的PC12细胞的影响,并探讨二型超氧化物歧化酶(Mn-SOD,SOD2)是否介导了BBR的保护作用。方法:将PC12细胞分为5组,分别为正常培养的对照组(Control)、25μM的6-OHDA损伤组、1μM的BBR预处理24 h组(BBR+6-OHDA)、SOD2-siRNA干扰组(SOD2-siRNA+BBR+6-OHDA)和乱序siRNA处理组(SC-siRNA+BBR+6-OHDA),孵育24 h后,采用噻唑蓝法(Methylthiazolyldiphenyl-tetrazolium bromide,MTT)检测细胞活力,试剂盒检测培养基乳酸脱氢酶(Lactic Dehydrogenase,LDH)、细胞内活性氧(Reactive Oxygen Species,ROS)、还原型谷胱甘肽(Glutathione,GSH)和过氧化氢酶(Catalase,CAT)的含量,使用流式细胞仪检测凋亡率,Western blot检测SOD2和凋亡蛋白Cleaved caspase-3的表达。结果:与Control组相比,6-OHDA诱导PC12细胞24 h后,细胞活力显著降低,SOD2表达、GSH和CAT的含量明显减少,培养基上清液LDH活力、细胞凋亡率、Cleaved caspase-3表达和ROS水平显著增加(P<0.05),而BBR预处理可显著恢复6-OHDA诱导的PC12细胞活力、SOD2表达、GSH和CAT水平,并降低细胞凋亡率、凋亡蛋白表达和细胞ROS水平(P<0.05),而SOD2-siRNA显著逆转了BBR预处理产生的上述保护作用(P<0.05),SC-siRNA则未对BBR预处理产生的上述作用造成明显影响(P>0.05)。结论:黄连素预处理可减轻6-OHDA诱导的PC12细胞损伤,而SOD2分子介导了BBR预处理对暴露于6-OHDA的PC12细胞的保护作用。     

Inhibition of Glutamate Transport in Synaptosomes by Dopamine Oxidation and Reactive Oxygen Species

Abstract: Dopamine can form reactive oxygen species and other reactive metabolites that can modify proteins and other cellular constituents. In this study, we tested the effect of dopamine oxidation products, other generators of reactive oxygen species, and a sulfhydryl modifier on the function of glutamate transporter proteins. We also compared any effects with those on the dopamine transporter, a protein whose function we had previously shown to be inhibited by dopamine oxidation. Preincubation with the generators of reactive oxygen species, ascorbate (0.85 m M ) or xanthine (500 µ M ) plus xanthine oxidase (25 mU/ml), inhibited the uptake of [³H]glutamate (10 µ M ) into rat striatal synaptosomes (−54 and −74%, respectively). The sulfhydryl-modifying agent N -ethylmaleimide (50–500 µ M ) also led to a dose-dependent inhibition of [³H]glutamate uptake. Preincubation with dopamine (100 µ M ) under oxidizing conditions inhibited [³H]glutamate uptake by 25%. Exposure of synaptosomes to increasing amounts of dopamine quinone by enzymatically oxidizing dopamine with tyrosinase (2–50 U/ml) further inhibited [³H]glutamate uptake, an effect prevented by the addition of glutathione. The effects of free radical generators and dopamine oxidation on [³H]glutamate uptake were similar to the effects on [³H]dopamine uptake (250 n M ). Our findings suggest that reactive oxygen species and dopamine oxidation products can modify glutamate transport function, which may have implications for neurodegenerative processes such as ischemia, methamphetamine-induced toxicity, and Parkinson's disease.