Neuroprotection with the Cannabidiol Quinone Derivative VCE-004.8 (EHP-101) against 6-Hydroxydopamine in Cell and Murine Models of Parkinson's Disease.

The 3-hydroxyquinone derivative of the non-psychotrophic phytocannabinoid cannabigerol, so-called VCE-003.2, and some other derivatives have been recently investigated for neuroprotective properties in experimental models of Parkinson's disease (PD) in mice. The pharmacological effects in those models were related to the activity on the peroxisome proliferator-activated receptor-γ (PPAR-γ) and possibly other pathways. In the present study, we investigated VCE-004.8 (formulated as EHP-101 for oral administration), the 3-hydroxyquinone derivative of cannabidiol (CBD), with agonist activity at the cannabinoid receptor type-2 (CB2) receptor in addition to its activity at the PPAR-γ receptor. Studies were conducted in both in vivo (lesioned-mice) and in vitro (SH-SY5Y cells) models using the classic parkinsonian neurotoxin 6-hydroxydopamine (6-OHDA). Our data confirmed that the treatment with VCE-004.8 partially reduced the loss of tyrosine hydroxylase (TH)-positive neurons measured in the substantia nigra of 6-OHDA-lesioned mice, in parallel with an almost complete reversal of the astroglial (GFAP) and microglial (CD68) reactivity occurring in this structure. Such neuroprotective effects attenuated the motor deficiencies shown by 6-OHDA-lesioned mice in the cylinder rearing test, but not in the pole test. Next, we explored the mechanism involved in the beneficial effect of VCE-004.8 in vivo, by analyzing cell survival in cultured SH-SY5Y cells exposed to 6-OHDA. We found an important cytoprotective effect of VCE-004.8 at a concentration of 10 µM, which was completely reversed by the addition of antagonists, T0070907 and SR144528, aimed at blocking PPAR-γ and CB2 receptors, respectively. The treatment with T0070907 alone only caused a partial reversal, whereas SR144528 alone had no effect, indicating a major contribution of PPAR-γ receptors in the cytoprotective effect of VCE-004.8 at 10 µM. In summary, our data confirmed the neuroprotective potential of VCE-004.8 in 6-OHDA-lesioned mice, and in vitro studies confirmed a greater relevance for PPAR-γ receptors rather than CB2 receptors in these effects.

pH-Responsive Redox Nanoparticles Protect SH-SY5Y Cells at Lowered pH in a Cellular Model of Parkinson's Disease.

The damage to SH-SY5Y cells by 6-hydroxydopamine (6-OHDA) is an established cellular model of Parkinson's disease (PD). Redox nanoparticles are a promising tool for therapy, including neurodegenerative diseases. As pH of the brain tissue at sites affected by PD is lowered down to 6.5, we studied the effect of pH-responsive redox nanoparticles (poly(ethylene glycol)-b-poly[4-(2,2,6,6-tetramethylpiperidine-1-oxyl)aminomethylstyrene]), which change their structure in a pH-dependent manner and become active below pH 7 (NRNPs pH), on the viability of SH-SY5Y cells treated with 6-OHDA at pH 6.5 and 7.4. Pretreatment of the cells with NRNPs pH (15-75 µM) prior to the 6-OHDA treatment increased their survival in a concentration-dependent manner at pH 6.5, but not at pH 7.4. Among several parameters studied (ATP and GSH content, the level of reactive oxygen species, mitochondrial potential, mitochondrial mass), only the mitochondrial mass was dose-dependently protected by NRNPs pH at pH 6.5, but not at pH 7.4. These results indicate that the action of NRNPs pH on mitochondria underlies their protective effect in this cellular model of PD. These results may have potential importance for future applications of NRNPs pH in preclinical and perhaps clinical studies.

Targeting ß-arrestin2 in the treatment of L-DOPA-induced dyskinesia in Parkinson's disease.

Parkinson's disease (PD) is characterized by severe locomotor deficits and is commonly treated with the dopamine (DA) precursor l-3,4-dihydroxyphenylalanine (L-DOPA), but its prolonged use causes dyskinesias referred to as L-DOPA-induced dyskinesias (LIDs). Recent studies in animal models of PD have suggested that dyskinesias are associated with the overactivation of G protein-mediated signaling through DA receptors. ß-Arrestins desensitize G protein signaling at DA receptors (D1R and D2R) in addition to activating their own G protein-independent signaling events, which have been shown to mediate locomotion. Therefore, targeting ß-arrestins in PD L-DOPA therapy might prove to be a desirable approach. Here we show in a bilateral DA-depletion mouse model of Parkinson's symptoms that genetic deletion of ß-arrestin2 significantly limits the beneficial locomotor effects while markedly enhancing the dyskinesia-like effects of acute or chronic L-DOPA treatment. Viral rescue or overexpression of ß-arrestin2 in knockout or control mice either reverses or protects against LIDs and its key biochemical markers. In other more conventional animal models of DA neuron loss and PD, such as 6-hydroxydopamine-treated mice or rats and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated nonhuman primates, ß-arrestin2 overexpression significantly reduced dyskinesias while maintaining the therapeutic effect of L-DOPA. Considerable efforts are being spent in the pharmaceutical industry to identify therapeutic approaches to block LIDs in patients with PD. Our results point to a potential therapeutic approach, whereby development of either a genetic or pharmacological intervention to enhance ß-arrestin2- or limit G protein-dependent D1/D2R signaling could represent a more mechanistically informed strategy.

Nitric oxide regulates synaptic transmission between spiny projection neurons.

Recurrent axon collaterals are a major means of communication between spiny projection neurons (SPNs) in the striatum and profoundly affect the function of the basal ganglia. However, little is known about the molecular and cellular mechanisms that underlie this communication. We show that intrastriatal nitric oxide (NO) signaling elevates the expression of the vesicular GABA transporter (VGAT) within recurrent collaterals of SPNs. Down-regulation of striatal NO signaling resulted in an attenuation of GABAergic signaling in SPN local collaterals, down-regulation of VGAT expression in local processes of SPNs, and impaired motor behavior. PKG1 and cAMP response element-binding protein are involved in the signal transduction that transcriptionally regulates VGAT by NO. These data suggest that transcriptional control of the vesicular GABA transporter by NO regulates GABA transmission and action selection.

Saturation Binding of Nicotine to Synthetic Neuromelanin Demonstrated by Fluorescence Spectroscopy.

Neuromelanin (NM) has long been considered as an aging pigment, perhaps an unavoidable and undesirable byproduct of dopaminergic neural transmission. However, NM is carefully packaged into double membrane-bound structures within cells of the substantia nigra and other neural tissues, suggesting a beneficial function to maintaining these stores. It is well established that NM is able to concentrate toxic xenobiotics within pigmented cells due to its unique chemical environment. In doing so, such agents may confer susceptibility to Parkinson's disease (PD) as illustrated by model PD-inducing neurotoxins such as methyl-phenyl-pyridinium ion. It is possible that high-affinity binding interactions toward NM may contribute to the adverse effects of PD-inducing toxins, as well as neuroprotective agents. Here we aim to develop a generalized assay capable of elucidating the binding constants of chemical agents to synthetic and natural neuromelanins. Toward this end, a model neuromelanin synthesized from dopamine and cysteine was prepared according to published procedure. Using a UV/Visible spectroscopic assay, we show that dopamine, 6-hydroxy dopamine, and nicotine bind to the synthetic neuromelanin, while caffeine did not. More importantly, nicotine was further found to induce a fluorescence signal in the presence of NM which was used to establish a binding constant estimated at 0.65 mM. Dopamine appears to enhance this signal, also in a saturable manner, with an estimated Kd of 0.05 mM in our isolated chemical system. In summary, the micro-scale fluorescence assay described herein will allow us to overcome many of the problems inherent in the study of chemical interaction with NM through traditional spectroscopic means. Using a single standardized signal, it should now be possible to rank a number of PD-related toxins based on NM-binding affinity and shed further light on this important problem.

Carnosic acid prevents 6-hydroxydopamine-induced cell death in SH-SY5Y cells via mediation of glutathione synthesis.

Understanding the neuroprotective effects of the rosemary phenolic diterpene carnosic acid (CA) has attracted increasing attention. We explored the mechanism by which CA modulates the neurotoxic effects of 6-hydroxydopamine (6-OHDA) in SH-SY5Y cells. Cells were pretreated with CA for 12 h followed by treatment with 100 µM 6-OHDA for 12 or 24 h. Cell viability determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolim bromide (MTT) assay indicated that 0.1 to 1 µM CA dose-dependently attenuated the cell death induced by 6-OHDA, whereas the effect of 3-5 µM CA was weaker. CA at 1 µM suppressed the 6-OHDA-induced nuclear condensation, reactive oxygen species generation, and cleavage of caspase 3 and PARP. Immunoblots showed that the phosphorylation of c-Jun NH(2)-terminal kinase (JNK) and p38 by 6-OHDA was reduced in the presence of CA. Incubation of cells with CA resulted in significant increases in the total glutathione (GSH) level and the protein expression of the γ-glutamylcysteine ligase catalytic subunit and modifier subunit. L-Buthionine-sulfoximine, an inhibitor of GSH synthesis, attenuated the effect of CA on cell death and apoptosis. Treatment with CA also led to an increase in nuclear factor erythroid-2 related factor 2 (Nrf2) activation, antioxidant response element (ARE)-luciferase reporter activity, and DNA binding to the ARE. Silencing of Nrf2 expression alleviated the reversal of p38 and JNK1/2 activation by CA. These results suggest that the attenuation of 6-OHDA-induced apoptosis by CA is associated with the Nrf2-driven synthesis of GSH, which in turn down-regulates the JNK and p38 signaling pathways. The CA compound may be a promising candidate for neuroprotection in Parkinson's disease.

Grafts Derived from an α-Synuclein Triplication Patient Mediate Functional Recovery but Develop Disease-Associated Pathology in the 6-OHDA Model of Parkinson's Disease.

BACKGROUND: Human induced pluripotent stem cells (hiPSCs) have been proposed as an alternative source for cell replacement therapy for Parkinson's disease (PD) and they provide the option of using the patient's own cells. A few studies have investigated transplantation of patient-derived dopaminergic (DA) neurons in preclinical models; however, little is known about the long-term integrity and function of grafts derived from patients with PD. OBJECTIVE: To assess the viability and function of DA neuron grafts derived from a patient hiPSC line with an α-synuclein gene triplication (AST18), using a clinical grade human embryonic stem cell (hESC) line (RC17) as a reference control. METHODS: Cells were differentiated into ventral mesencephalic (VM)-patterned DA progenitors using an established GMP protocol. The progenitors were then either terminally differentiated to mature DA neurons in vitro or transplanted into 6-hydroxydopamine (6-OHDA) lesioned rats and their survival, maturation, function, and propensity to develop α-synuclein related pathology, were assessed in vivo. RESULTS: Both cell lines generated functional neurons with DA properties in vitro. AST18-derived VM progenitor cells survived transplantation and matured into neuron-rich grafts similar to the RC17 cells. After 24 weeks, both cell lines produced DA-rich grafts that mediated full functional recovery; however, pathological changes were only observed in grafts derived from the α-synuclein triplication patient line. CONCLUSION: This data shows proof-of-principle for survival and functional recovery with familial PD patient-derived cells in the 6-OHDA model of PD. However, signs of slowly developing pathology warrants further investigation before use of autologous grafts in patients.

A functionalized hydroxydopamine quinone links thiol modification to neuronal cell death.

Recent findings suggest that dopamine oxidation contributes to the development of Parkinson's disease (PD); however, the mechanistic details remain elusive. Here, we compare 6-hydroxydopamine (6-OHDA), a product of dopamine oxidation that commonly induces dopaminergic neurodegeneration in laboratory animals, with a synthetic alkyne-functionalized 6-OHDA variant. This synthetic molecule provides insights into the reactivity of quinone and neuromelanin formation. Employing Huisgen cycloaddition chemistry (or "click chemistry") and fluorescence imaging, we found that reactive 6-OHDA p-quinones cause widespread protein modification in isolated proteins, lysates and cells. We identified cysteine thiols as the target site and investigated the impact of proteome modification by quinones on cell viability. Mass spectrometry following cycloaddition chemistry produced a large number of 6-OHDA modified targets including proteins involved in redox regulation. Functional in vitro assays demonstrated that 6-OHDA inactivates protein disulfide isomerase (PDI), which is a central player in protein folding and redox homeostasis. Our study links dopamine oxidation to protein modification and protein folding in dopaminergic neurons and the PD model.

Preparation and evaluation of injectable Rasagiline mesylate dual-controlled drug delivery system for the treatment of Parkinson's disease.

A microsphere-gel in situ forming implant (MS-Gel ISFI) dual-controlled drug delivery system was applied to a high water-soluble small-molecule compound Rasagiline mesylate (RM) for effective treatment of Parkinson's disease. This injectable complex depot system combined an in situ phase transition gel with high drug-loading and encapsulation efficiency RM-MS prepared by a modified emulsion-phase separation method and optimized by Box-Behnken design. It was evaluated for in vitro drug release, in vivo pharmacokinetics, and in vivo pharmacodynamics. We found that the RM-MS-Gel ISFI system showed no initial burst release and had a long period of in vitro drug release (60 days). An in vivo pharmacokinetic study indicated a significant reduction (p < .01) in the initial high plasma drug concentration of the RM-MS-Gel ISFI system compared to that of the single RM-MS and RM-in situ gel systems after intramuscular injection to rats. A pharmacodynamic study demonstrated a significant reduction (p < .05) in 6-hydroxydopamine-induced contralateral rotation behavior and an effective improvement (p < .05) in dopamine levels in the striatum of the lesioned side after 28 days in animals treated with the RM-MS-Gel ISFI compared with that of animals treated with saline. MS-embedded in situ phase transition gel is superior for use as a biodegradable and injectable sustained drug delivery system with a low initial burst and long period of drug release for highly hydrophilic small molecule drugs.

Dimerumic Acid and Deferricoprogen Activate Ak Mouse Strain Thymoma/Heme Oxygenase-1 Pathways and Prevent Apoptotic Cell Death in 6-Hydroxydopamine-Induced SH-SY5Y Cells.

Parkinson's disease (PD) is a neurodegenerative disorder, which can be modeled using the neurotoxin 6-hydroxydopamine (6-OHDA) to generate oxidative stress. Here, we studied the effects of the antioxidants deferricoprogen (DFC) and dimerumic acid (DMA), produced by rice fermented with Monascus purpureus NTU 568, on 6-OHDA-induced apoptosis in SH-SY5Y cells and their potential protective mechanisms. DMA and DFC inhibited 6-OHDA-induced apoptosis and cellular reactive oxygen species (ROS) in SH-SY5Y human neuroblastoma cells. Molecular analysis demonstrated associated upregulation of the Ak mouse strain thymoma (Akt), heme oxygenase-1 (HO-1), and signal-regulated kinase (ERK) pathways along with inhibited phosphorylation of c-Jun N-terminal kinase (JNK) and p38 pathways and altered homodimeric glycoprotein, N-methyl-d-aspartate (NMDA) receptor, and immunoglobulin Fc receptor gene expression. These results suggested that the neuroprotection elicited by DMA and DFC against 6-OHDA-induced neurotoxicity was associated with the Akt, MAPK, and HO-1 pathways via regulating the gene expression of NMDA receptor, homodimeric glycoprotein, and immunoglobulin Fc receptor.

Allogeneic/xenogeneic transplantation of peptide-labeled mitochondria in Parkinson's disease: restoration of mitochondria functions and attenuation of 6-hydroxydopamine-induced neurotoxicity.

Although restoration of mitochondrial function in mitochondrial diseases through peptide-mediated allogeneic mitochondrial delivery (PMD) has been demonstrated in vitro, the in vivo therapeutic efficacy of PMD in Parkinson's disease (PD) has yet to be determined. In this study, we compared the functionality of mitochondrial transfer with or without Pep-1 conjugation in neurotoxin (6-hydroxydopamine, 6-OHDA)-induced PC12 cells and PD rat models. We injected mitochondria into the medial forebrain bundle (MFB) of the PD rats after subjecting the nigrostriatal pathway to a unilateral 6-OHDA lesion for 21 days, and we verified the effectiveness of the mitochondrial graft in enhancing mitochondrial function in the soma of the substantia nigra (SN) neuron through mitochondrial transport dynamics in the nigrostriatal circuit. The result demonstrated that only PMD with allogeneic and xenogeneic sources significantly sustained mitochondrial function to resist the neurotoxin-induced oxidative stress and apoptotic death in the rat PC12 cells. The remaining cells exhibited a greater capability of neurite outgrowth. Furthermore, allogeneic and xenogeneic transplantation of peptide-labeled mitochondria after 3 months improved the locomotive activity in the PD rats. This increase was accompanied by a marked decrease in dopaminergic neuron loss in the substantia nigra pars compacta (SNc) and consistent enhancement of tyrosine hydroxylase-positive immunoreaction of dopaminergic neurons in the SNc and striatum. We also observed that in the SN dopaminergic neuron in the treated PD rats, mitochondrial complex I protein and mitochondrial dynamics were restored, thus ameliorating the oxidative DNA damage. Moreover, we determined signal translocation of graft allogeneic mitochondria from the MFB to the calbindin-positive SN neuron, which demonstrated the regulatory role of mitochondrial transport in alleviating 6-OHDA-induced degeneration of dopaminergic neurons.

The effects of glutathione and ascorbic acid on the oxidations of 6-hydroxydopa and 6-hydroxydopamine.

The interactions of ascorbic acid (AA) and reduced glutathione (GSH) in the oxidations of the catecholaminergic neurotoxins 6-hydroxydopa (TOPA) and 6-hydroxydopamine (6-OHDA) were investigated by both high performance liquid chromatography with electrochemical detection (HPLC-ED) and spectrometric methods. These comparative studies showed TOPA and 6-OHDA to be extremely unstable, with 100% of the trihydroxyphenyls oxidized within 0.5 min at physiological pH in potassium phosphate buffer. Neither AA nor GSH was found capable of significantly impeding the oxidations of these trihydroxyphenyls, or of regenerating these substances by reducing back their oxidation products, even though such a redox exchange mechanism was demonstrated for AA and the dihydroxyphenyl dopamine. Although ineffective in keeping TOPA and 6-OHDA as reduced molecules, GSH may nevertheless influence the neurotoxicity of trihydroxyphenyls by interacting with their oxidation products forming glutathionyl conjugates, thereby switching the reaction pathway away from potentially toxic eumelanin precursors and toward the production of pheomelanin. Electrochemical analyses established the formation of two oxidation products derived from each trihydroxyphenyl, one detected at -100 mV and the other at +700 mV. AA had no effect on either oxidation product, whereas GSH significantly decreased the levels of both oxidation products. The component detected at +700 mV is the cyclized, reduced leukochrome. The identity of the component detected at -100 mV was not established, but it is considered to be either the p-quinone or the cyclized, oxidized aminochrome.

Systemic human CD34(+) cells populate the brain and activate host mechanisms to counteract nigrostriatal degeneration.

AIM: Here we investigated the neuroprotective potential of systemic CD34(+) human cord blood cells (hCBCs) in a 6-hydroxydopamine rat model of Parkinson's disease. METHODS: Purified CD34(+) hCBCs were intravenously administered to rats subjected to 6-hydroxydopamine 24 h earlier, and behavioral and immunohistological analysis performed. RESULTS: CD34(+) hCBC administration significantly prevented host nigrostriatal degeneration inducing behavioral recovery in treated rats. Although donor hCBCs did not differentiate into neural phenotypes, they stimulated the production of new neuroblasts and angiogenesis, and reduced gliosis in recipient animals. Importantly, surviving donor hCBCs were identified, and their tissue distribution pattern correlated with the observed therapeutic effects. CONCLUSION: Peripherally applied CD34(+) hCBCs can migrate into brain tissues and elicit host-based protective mechanisms to support the survival of midbrain dopamine neurons.

Untangling dopamine-adenosine receptor-receptor assembly in experimental parkinsonism in rats.

Parkinson's disease (PD) is a dopaminergic-related pathology in which functioning of the basal ganglia is altered. It has been postulated that a direct receptor-receptor interaction - i.e. of dopamine D2 receptor (D2R) with adenosine A2A receptor (A2AR) (forming D2R-A2AR oligomers) - finely regulates this brain area. Accordingly, elucidating whether the pathology prompts changes to these complexes could provide valuable information for the design of new PD therapies. Here, we first resolved a long-standing question concerning whether D2R-A2AR assembly occurs in native tissue: by means of different complementary experimental approaches (i.e. immunoelectron microscopy, proximity ligation assay and TR-FRET), we unambiguously identified native D2R-A2AR oligomers in rat striatum. Subsequently, we determined that, under pathological conditions (i.e. in a rat PD model), D2R-A2AR interaction was impaired. Collectively, these results provide definitive evidence for alteration of native D2R-A2AR oligomers in experimental parkinsonism, thus conferring the rationale for appropriate oligomer-based PD treatments.

Parkinson's disease and enhanced inflammatory response.

Parkinson's disease (PD) is the first and second most prevalent motor and neurodegenerative disease, respectively. The clinical symptoms of PD result from a loss of midbrain dopaminergic (DA) neurons. However, the molecular cause of DA neuron loss remains elusive. Mounting evidence implicates enhanced inflammatory response in the development and progression of PD pathology. This review examines current research connecting PD and inflammatory response.

Hydroxyl radical reactions and the radical scavenging activity of ß-carboline alkaloids.

ß-Carbolines are bioactive pyridoindole alkaloids occurring in foods, plants and the human body. Their activity as hydroxyl radical (OH) scavengers is reported here by using three different methods: deoxyribose degradation, hydroxylation of benzoate and hydroxylation of 2'-deoxyguanosine to give 8-hydroxy-2'-deoxyguanosine (8-OHdG) as assessed by RP-HPLC (MS). Fenton reactions (Fe(2+)/Fe(3+) plus H2O2) were used for OH generation, and the radical increased in the presence of ascorbic acid or 6-hydroxydopamine as pro-oxidants. ß-Carbolines were scavengers of OH in the three assays and in the presence of pro-oxidants. Tetrahydro-ß-carboline-3-carboxylic acids were active against the hydroxylation of 2'-deoxyguanosine. ß-Carbolines reacted with hydroxyl radicals (OH) affording hydroxy-ß-carbolines, whereas tetrahydro-ß-carbolines gave oxidative and degradation products. On the basis of IC50 and reaction rates (k), ß-carbolines (norharman and harman), and tetrahydro-ß-carbolines (tetrahydro-ß-carboline, 1-methyltetrahydro-ß-carboline and pinoline) were good OH radical scavengers and their activity was comparable to that of the indole, melatonin, which is an effective hydroxyl radical scavenger and antioxidant.

Trans-blood brain barrier delivery of dopamine-loaded nanoparticles reverses functional deficits in parkinsonian rats.

Sustained and safe delivery of dopamine across the blood brain barrier (BBB) is a major hurdle for successful therapy in Parkinson's disease (PD), a neurodegenerative disorder. Therefore, in the present study we designed neurotransmitter dopamine-loaded PLGA nanoparticles (DA NPs) to deliver dopamine to the brain. These nanoparticles slowly and constantly released dopamine, showed reduced clearance of dopamine in plasma, reduced quinone adduct formation, and decreased dopamine autoxidation. DA NPs were internalized in dopaminergic SH-SY5Y cells and dopaminergic neurons in the substantia nigra and striatum, regions affected in PD. Treatment with DA NPs did not cause reduction in cell viability and morphological deterioration in SH-SY5Y, as compared to bulk dopamine-treated cells, which showed reduced viability. Herein, we report that these NPs were able to cross the BBB and capillary endothelium in the striatum and substantia nigra in a 6-hydroxydopamine (6-OHDA)-induced rat model of PD. Systemic intravenous administration of DA NPs caused significantly increased levels of dopamine and its metabolites and reduced dopamine-D2 receptor supersensitivity in the striatum of parkinsonian rats. Further, DA NPs significantly recovered neurobehavioral abnormalities in 6-OHDA-induced parkinsonian rats. Dopamine delivered through NPs did not cause additional generation of ROS, dopaminergic neuron degeneration, and ultrastructural changes in the striatum and substantia nigra as compared to 6-OHDA-lesioned rats. Interestingly, dopamine delivery through nanoformulation neither caused alterations in the heart rate and blood pressure nor showed any abrupt pathological change in the brain and other peripheral organs. These results suggest that NPs delivered dopamine into the brain, reduced dopamine autoxidation-mediated toxicity, and ultimately reversed neurochemical and neurobehavioral deficits in parkinsonian rats.

Roles of phosphate and an enoyl radical in ferritin iron mobilization by 5-aminolevulinic acid.

5-Aminolevulinic acid (ALA), a heme precursor that accumulates in acute intermittent porphyria (AIP) and lead poisoning, undergoes enolization and subsequent iron-catalyzed oxidation at neutral pH. Iron is released from horse spleen ferritin (HoSF) by both ALA-generated O(2)(.-) and enoyl radical (ALA(z.rad)), which amplifies the chain of ALA oxidation (autocatalysis). Iron chelators such as EDTA, ATP, but not citrate, and phosphate accelerate this process and ALA-promoted iron release from HoSF is faster in horse spleen isoferritins containing larger amounts of phosphate in the core. ALA (+0.377 V versus standard hydrogen electrode) is less effective in releasing iron from ferritin than are thioglycollic acid, 6-hydroxydopamine, and N,N,N', N'-tetramethyl-p-phenylenediamine. During electrochemical one electron oxidation of ALA in a nitrogen atmosphere, spin trapping experiments with 3,5-dibromo-4-nitrosobenzenesulfonic acid demonstrated the formation of a spin adduct characterized by a six line signal, indicating a secondary carbon-centered radical and attributed to a resonant ALA&z.rad; radical. Iron is also released in such anaerobic electrochemical oxidations of ALA in the presence of ferritin, suggesting that, in addition to O(2)(*-), ALA&z.rad; can promote iron mobilization from ferritin. Hence, ALA&z.rad; may amplify the metal-catalyzed oxidation of ALA, damaging ALA-accumulating cells and possibly contributing to the symptoms of porphyria.

Generation of the neurotoxin 6-hydroxydopamine by peroxidase/H2O2 oxidation of dopamine.

At physiological pH values, oxidation of the neurotransmitter dopamine (DA) by the peroxidase/H2O2 system leads to, besides dopaminochrome and 5,6-dihydroxyindole resulting from oxidative cyclization of dopaminequinone (DQ), significant amounts of the neurotoxin 6-hydroxydopamine (6-OHDA) in the oxidized quinonoid form (topaminequinone, TQ). Formation of TQ was shown to depend critically on the presence of hydrogen peroxide in the reaction medium and was not observed when DA oxidation was carried out using the tyrosinase/O2 system or chemical agents such as periodate or ferricyanide. These and other data suggest that, under the conditions adopted, nucleophilic attack of the hydrogen peroxide anion on DQ leading to TQ significantly competes with the intramolecular cyclization path. In line with this mechanism, the reaction course was not affected by the presence of hydroxyl radical scavengers. Peroxidase/H2O2 oxidation of the model N-acetyldopamine (1) gave, as expected, the 2-hydroxy-1,4-benzoquinone 3 in yields up to 55%, depending on the catecholamine/H2O2 mole ratio. Likewise, reaction of 4-methyl-1,2-benzoquinone (4) with hydrogen peroxide afforded 2-hydroxy-5-methyl-1,4-benzoquinone (5) in good yields. Collectively, these results would point to the possibility that intraneuronal formation of 6-OHDA is associated with an increased production of hydrogen peroxide under oxidative stress conditions.

Modulation of 6-hydroxydopamine oxidation by various proteins.

The spontaneous autoxidation of the neurotoxin 6-hydroxydopamine proceeds by a free radical chain reaction involving the superoxide anion radical and produces the corresponding chromogen 6-hydroxydopamine quinone and hydrogen peroxide. The rate of this reaction is increased in the presence of ceruloplasmin and peroxidase, and reduced by superoxide dismutase, catalase, and DT-diaphorase. We report some explanations of why these proteins may increase or reduce the rate of autoxidation of 6-hydroxydopamine.