Juan Segura-Aguilar

Quercetin may act as a cytotoxic prooxidant after its metabolic activation to semiquinone and quinoidal product

In the last ten years, there has been an important increase in interest in quercetin action as a unique antioxidant, but its putative role in numerous prooxidant effects is also being continually updated. The mechanism underlying this undesirable ability seems to involve its metabolic oxidoreductive activation. Based on the structural properties of quercetin, we have investigated whether its catechol moiety may be the potential tool for revealed toxicity. We demonstrated, with an ESR spin-stabilization technique coupled to conventional spectrophotometry, that o-semiquinone and o-quinone are indeed the products of enzymatically catalyzed oxidative degradation of quercetin. The former radical might serve to facilitate the formation of superoxide and depletion of GSH, which could confer a specificity of its prooxidative action in situ. The observed one-electron reduction of o-quinone may enrich the semiquinone pool, thereby magnifying its effect. The two-electron reduction of quinone can result in constant resupply of quercetin in situ, thereby also modulating another pathway of its known biological activities. We have also tried to see whether the intracellular oxidative degradation of quercetin can be confirmed under the controlled conditions of model monolayer cell cultures. The results are indicative of the intracellular metabolic activation of quercetin to o-quinone, the process which can be partially associated with the observed concentration-dependent cytotoxic effect of quercetin.

On the mechanism of the Mn3(+)-induced neurotoxicity of dopamine:prevention of quinone-derived oxygen toxicity by DT diaphorase and superoxide dismutase.

Dopamine (DA) is rapidly oxidized by Mn3(+)-pyrophosphate to its cyclized o-quinone (cDAoQ), a reaction which can be prevented by NADH, reduced glutathione (GSH) or ascorbic acid. The oxidation of DA by Mn3+, which appears to be irreversible, results in a decrease in the level of DA, but not in a formation of reactive oxygen species, since oxygen is neither consumed nor required in this reaction. The formation of cDAoQ can initiate the generation of superoxide radicals (O2-.) by reduction-oxidation cycling, i.e. one-electron reduction of the quinone by various NADH- or NADPH-dependent flavoproteins to the semiquinone (QH.), which is readily reoxidized by O2 with the concomitant formation of O2-.. This mechanism is believed to underly the cytotoxicity of many quinones. Two-electron reduction of cDAoQ to the hydroquinone can be catalyzed by the flavoprotein DT diaphorase (NAD(P)H:quinone oxidoreductase). This enzyme efficiently maintains DA quinone in its fully reduced state, although some reoxidation of the hydroquinone (QH2) is observed (QH2 + O2----QH. + O2-. + H+; QH. + O2----Q + O2-.). In the presence of Mn3+, generated from Mn2+ by O2-. (Mn2+ + 2H+ + O2-.----Mn3+ + H2O2) formed during the autoxidation of DA hydroquinone, the rate of autoxidation is increased dramatically as is the formation of H2O2. Furthermore, cDAoQ is no longer fully reduced and the steady-state ratio between the hydroquinone and the quinone is dependent on the amount of DT diaphorase present. The generation of Mn3+ is inhibited by superoxide dismutase (SOD), which catalyzes the disproportionation of O2-. to H2O2 and O2. It is noteworthy that addition of SOD does not only result in a decrease in the amount of H2O2 formed during the regeneration of Mn3+, but, in fact, prevents H2O2 formation. Furthermore, in the presence of this enzyme the consumption of O2 is low, as is the oxidation of NADH, due to autoxidation of the hydroquinone, and the cyclized DA o-quinone is found to be fully reduced. These observations can be explained by the newly-discovered role of SOD as a superoxide:semiquinone (QH.) oxidoreductase catalyzing the following reaction: O2-. + QH. + 2H+----QH2 + O2. Thus, the combination of DT diaphorase and SOD is an efficient system for maintaining cDAoQ in its fully reduced state, a prerequisite for detoxication of the quinone by conjugation with sulfate or glucuronic acid. In addition, only minute amounts of reactive oxygen species will be formed, i.e. by the generation of O2-., which through disproportionation to H2O2 and further reduction by ferrous ions can be converted to the hydroxyl radical (OH.). Absence or low levels of these enzymes may create an oxidative stress on the cell and thereby initiate events leading to cell death.

Targeting the UPR transcription factor XBP1 protects against Huntington's disease through the regulation of FoxO1 and autophagy

Mutations leading to expansion of a poly-glutamine track in Huntingtin (Htt) cause Huntingtons disease (HD). Signs of endoplasmic reticulum (ER) stress have been recently reported in animal models of HD, associated with the activation of the unfolded protein response (UPR). Here we have investigated the functional contribution of ER stress to HD by targeting the expression of two main UPR transcription factors, XBP1 and ATF4 (activating transcription factor 4), in full-length mutant Huntingtin (mHtt) transgenic mice. XBP1-deficient mice were more resistant to developing disease features, associated with improved neuronal survival and motor performance, and a drastic decrease in mHtt levels. The protective effects of XBP1 deficiency were associated with enhanced macroautophagy in both cellular and animal models of HD. In contrast, ATF4 deficiency did not alter mHtt levels. Although, XBP1 mRNA splicing was observed in the striatum of HD transgenic brains, no changes in the levels of classical ER stress markers were detected in symptomatic animals. At the mechanistic level, we observed that XBP1 deficiency led to augmented expression of Forkhead box O1 (FoxO1), a key transcription factor regulating autophagy in neurons. In agreement with this finding, ectopic expression of FoxO1 enhanced autophagy and mHtt clearance in vitro. Our results provide strong evidence supporting an involvement of XBP1 in HD pathogenesis probably due to an ER stress-independent mechanism involving the control of FoxO1 and autophagy levels.

Protective and toxic roles of dopamine in Parkinson's disease

The molecular mechanisms causing the loss of dopaminergic neurons containing neuromelanin in the substantia nigra and responsible for motor symptoms of Parkinsons disease are still unknown. The discovery of genes associated with Parkinsons disease (such as alpha synuclein (SNCA), E3 ubiquitin protein ligase (parkin), DJ‐1 (PARK7), ubiquitin carboxyl‐terminal hydrolase isozyme L1 (UCHL‐1), serine/threonine‐protein kinase (PINK‐1), leucine‐rich repeat kinase 2 (LRRK2), cation‐transporting ATPase 13A1 (ATP13A), etc.) contributed enormously to basic research towards understanding the role of these proteins in the sporadic form of the disease. However, it is generally accepted by the scientific community that mitochondria dysfunction, alpha synuclein aggregation, dysfunction of protein degradation, oxidative stress and neuroinflammation are involved in neurodegeneration. Dopamine oxidation seems to be a complex pathway in which dopamine o‐quinone, aminochrome and 5,6‐indolequinone are formed. However, both dopamine o‐quinone and 5,6‐indolequinone are so unstable that is difficult to study and separate their roles in the degenerative process occurring in Parkinsons disease. Dopamine oxidation to dopamine o‐quinone, aminochrome and 5,6‐indolequinone seems to play an important role in the neurodegenerative processes of Parkinsons disease as aminochrome induces: (i) mitochondria dysfunction, (ii) formation and stabilization of neurotoxic protofibrils of alpha synuclein, (iii) protein degradation dysfunction of both proteasomal and lysosomal systems and (iv) oxidative stress. The neurotoxic effects of aminochrome in dopaminergic neurons can be inhibited by: (i) preventing dopamine oxidation of the transporter that takes up dopamine into monoaminergic vesicles with low pH and dopamine oxidative deamination catalyzed by monoamino oxidase (ii) dopamine o‐quinone, aminochrome and 5,6‐indolequinone polymerization to neuromelanin and (iii) two‐electron reduction of aminochrome catalyzed by DT‐diaphorase. Furthermore, dopamine conversion to NM seems to have a dual role, protective and toxic, depending mostly on the cellular context.

Copper neurotoxicity is dependent on dopamine-mediated copper uptake and one-electron reduction of aminochrome in a rat substantia nigra neuronal cell line.

The mechanism of copper (Cu) neurotoxicity was studied in the RCSN‐3 neuronal dopaminergic cell line, derived from substantia nigra of an adult rat. The formation of a Cu–dopamine complex was accompanied by oxidation of dopamine to aminochrome. We found that the Cu–dopamine complex mediates the uptake of 64CuSO4 into the Raúl Caviedes substantia nigra‐clone 3 (RCSN3) cells, and it is inhibited by the addition of excess dopamine (2 m m) (63%, p < 0.001) and nomifensine (2 µm) (77%, p < 0.001). Copper sulfate (1 m m) alone was not toxic to RCSN‐3 cells, but was when combined with dopamine or with dicoumarol (95% toxicity; p < 0.001) which inhibits DPNH and TPNH (DT)‐diaphorase. Electron spin resonance (ESR) spectrum of the 5,5‐dimethylpyrroline‐N‐oxide (DMPO) spin trap adducts showed the presence of a C‐centered radical when incubating cells with dopamine, CuSO4 and dicoumarol. A decrease in the expression of CuZn‐superoxide dismutase and glutathione peroxidase mRNA was observed when RCSN‐3 cells were treated with CuSO4, dopamine, or CuSO4 and dopamine. However, the mRNA expression of glutathione peroxidase remained at control levels when the cells were treated with CuSO4, dopamine and dicoumarol. The regulation of catalase was different since all the treatments with CuSO4 increased the expression of catalase mRNA. Our results suggest that copper neurotoxicity is dependent on: (i) the formation of Cu–dopamine complexes with concomitant dopamine oxidation to aminochrome; (ii) dopamine‐dependent Cu uptake; and (iii) one‐electron reduction of aminochrome.

Human Class Mu Glutathione Transferases, in Particular Isoenzyme M2-2, Catalyze Detoxication of the Dopamine Metabolite Aminochrome

Human glutathione transferases (GSTs) were shown to catalyze the reductive glutathione conjugation of aminochrome (2,3-dihydroindole-5,6-dione). The class Mu enzyme GST M2-2 displayed the highest specific activity (148 μmol/min/mg), whereas GSTs A1-1, A2-2, M1-1, M3-3, and P1-1 had markedly lower activities (<1 μmol/min/mg). The product of the conjugation, with a UV spectrum exhibiting absorption peaks at 277 and 295 nm, was 4-S-glutathionyl-5,6-dihydroxyindoline as determined by NMR spectroscopy. In contrast to reduced forms of aminochrome (leucoaminochrome and o-semiquinone), 4-S-glutathionyl-5,6-dihydroxyindoline was stable in the presence of molecular oxygen, superoxide radicals, and hydrogen peroxide. However, the strongly oxidizing complex of Mn3+ and pyrophosphate oxidizes 4-S-glutathionyl-5,6-dihydroxyindoline to 4-S-glutathionylaminochrome, a new quinone derivative with an absorption peak at 620 nm. GST M2-2 (and to a lower degree, GST M1-1) prevents the formation of reactive oxygen species linked to one-electron reduction of aminochrome catalyzed by NADPH-cytochrome P450 reductase. The results suggest that the reductive conjugation of aminochrome catalyzed by GSTs, in particular GST M2-2, is an important cellular antioxidant activity preventing the formation of o-semiquinone and thereby the generation of reactive oxygen species.

Interactions of iron, dopamine and neuromelanin pathways in brain aging and Parkinson's disease.

HIGHLIGHTSIn this review the multiways interactions/modulations between iron, dopamine oxidation, neuromelanin and their role in brain are discussed.The relationship between iron and dopamine oxidation with consequences on cells is presented.An overview of synthesis and properties of peripheral melanins is given.Neuromelanin synthesis, structure and interaction with iron are reviewed, considering their protective/toxic pathways in neurons.The role of iron and neuromelanin and their possible detrimental effects in brain aging and Parkinsons disease are appraised. ABSTRACT There are several interrelated mechanisms involving iron, dopamine, and neuromelanin in neurons. Neuromelanin accumulates during aging and is the catecholamine‐derived pigment of the dopamine neurons of the substantia nigra and norepinephrine neurons of the locus coeruleus, the two neuronal populations most targeted in Parkinsons disease. Many cellular redox reactions rely on iron, however an altered distribution of reactive iron is cytotoxic. In fact, increased levels of iron in the brain of Parkinsons disease patients are present. Dopamine accumulation can induce neuronal death; however, excess dopamine can be removed by converting it into a stable compound like neuromelanin, and this process rescues the cell. Interestingly, the main iron compound in dopamine and norepinephrine neurons is the neuromelanin‐iron complex, since neuromelanin is an effective metal chelator. Neuromelanin serves to trap iron and provide neuronal protection from oxidative stress. This equilibrium between iron, dopamine, and neuromelanin is crucial for cell homeostasis and in some cellular circumstances can be disrupted. Indeed, when neuromelanin‐containing organelles accumulate high load of toxins and iron during aging a neurodegenerative process can be triggered. In addition, neuromelanin released by degenerating neurons activates microglia and the latter cause neurons death with further release of neuromelanin, then starting a self‐propelling mechanism of neuroinflammation and neurodegeneration. Considering the above issues, age‐related accumulation of neuromelanin in dopamine neurons shows an interesting link between aging and neurodegeneration.

DT-Diaphorase maintains the reduced state of ubiquinones in lipid vesicles thereby promoting their antioxidant function.

The activity of purified DT-diaphorase in the reduction of ubiquinone homologues of different side-chain length incorporated in uni- and multilamellar vesicles was determined. The direct relationship between the reduced state of ubiquinones and the inhibition of lipid autoxidation induced by thermolabile azocompounds was also demonstrated. Results demonstrate that DT-diaphorase is able to generate and to maintain the reduced, antioxidant form of ubiquinones in both types of vesicles. Furthermore, the results reported herein show that, in the presence of nicotinamide adenine dinucleotide (NADH) and DT-diaphorase, ubiquinol-containing multilamellar vesicles exposed to a lipophilic azocompound did not undergo lipid peroxidation, whereas in vesicles lacking either NADH or DT-diaphorase, thiobarbituric acid reactive substances (TBARS) formation occurred. It is suggested that DT-diaphorase may be responsible for maintaining the reduced state of ubiquinones in various nonmitochondrial cellular membranes.

Effect of superoxide dismutase on the autoxidation of various hydroquinones. A possible role of superoxide dismutase as a superoxide: semiquinone oxidoreductase

The autoxidation of DT-diaphorase-reduced 1,4-naphthoquinone, 2-OH-1,4-naphthoquinone, and 2-OH-p-benzoquinone is efficiently prevented by superoxide dismutase. This effect was assessed in terms of an inhibition of NADPH oxidation (over the amount required to reduce the available quinone), O2 consumption, and H2O2 formation. Superoxide dismutase also affects the distribution of molecular products -hydroquinone/quinone-involved in autoxidation, by favoring the accumulation of the reduced form of the above quinones. In contrast, the rate of autoxidation of DT-diaphorase-reduced 1,2-naphthoquinone is enhanced by superoxide dismutase, as shown by increased rates of NADPH oxidation, O2 consumption, and H2O2 formation and by an enhanced accumulation of the oxidized product, 1,2-naphthoquinone. These findings suggest that superoxide dismutase can either prevent or enhance hydroquinone autoxidation. The former process would imply a possible new activity displayed by superoxide dismutase involving the reduction of a semiquinone by O2-.. This activity is probably restricted to the redox properties of the semiquinones under study, as indicated by the failure of superoxide dismutase to prevent autoxidation of 1,2-naphthohydroquinone.

Neurotoxins and neurotoxic species implicated in neurodegeneration

Neurotoxins, in the general sense, represent novel chemical structures which when administered in vivo orin vitro, are capable of producing neuronal damage or neurodegeneration—with some degree of specificity relating to neuronal phenotype or populations of neurons with specific characteristics (i.e., receptor type, ion channel type, astrocyte-dependence, etc.). The broader term ‘neurotoxin’ includes this categorization but extends the term to include intra- or extracellular mediators involved in the neurodegenerative event, including necrotic and apoptotic factors. Moreover, as it is recognized that astrocytes are essential supportive satellite cells for neurons, and because damage to these cells ultimately affects neuronal function, the term ‘neurotoxin’ might reasonably be extended to include those chemical species which also adversely affect astrocytes. This review is intended to highlight developments that have occurred in the field of ‘neurotoxins’ during the past 5 years, including MPTP/MPP+, 6-hydroxydopamine (6-OHDA), meth-amphetamine; salsolinol; leukoaminochrome-o-semi-quinone; rotenone; iron; paraquat; HPP+; veratridine; soman; glutamate; kainate; 3-nitropropionic acid; peroxynitrite anion; and metals (copper, manganese, lead, mercury). Neurotoxins represent tools to help elucidate intra- and extra-cellular processes involved in neuronal necrosis and apoptosis, so that drugs can be developed towards targets that interrupt the processes leading towards neuronal death.