Potential bioactivation pathways for the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

The metabolism of the selective nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been studied in rat brain mitochondrial incubation mixtures. The 1-methyl-4-phenylpyridinium species MPP+ has been characterized by chemical ionization mass spectral and 1H NMR analysis. Evidence also was obtained for the formation of an intermediate product which, with the aid of deuterium incorporation studies, was tentatively identified as the alpha-carbon oxidation product, the 1-methyl-4-phenyl-2,3-dihydropyridinium species MPDP+. Comparison of the diode array UV spectrum of this metabolite with that of the synthetic perchlorate salt of MPDP+ confirmed this assignment. The oxidation of MPTP to MPDP+ but not of MPDP+ to MPP+ is completely inhibited by 10(-7) M pargyline. MPDP+, on the other hand, is unstable and rapidly undergoes disproportionation to MPTP and MPP+. Based on these results, we speculate that the neurotoxicity of MPTP is mediated by its intraneuronal oxidation to MPDP+, a reaction which appears to be catalyzed by MAO. The interactions of MPDP+ and/or MPP+ with dopamine, a readily oxidizable compound present in high concentration in the nigrostriatum, to form neurotoxic species may account for the selective toxic properties of the parent drug.     

Role of 1-methyl-4-phenylpyridinium ion formation and accumulation in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity to isolated hepatocytes

The parkinsonian-inducing compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is converted by isolated hepatocytes to its primary metabolite, the 1-methyl-4-phenyl-2,3-dihydropyridinium ion (MPDP+), and to its fully oxidized derivative, 1-methyl-4-phenylpyridinium ion (MPP+). Only the latter, however, accumulates in the cells. Incubation of hepatocytes in the presence of MPDP+ also results in the selective intracellular accumulation of MPP+. Conversion to MPP+ is more rapid and extensive after exposure to MPDP+, than with MPTP and the former is also more toxic. Addition of MPP+ itself is toxic to hepatocytes but only after a long lag period, which presumably reflects its limited access to the cell and its relatively slow intracellular accumulation. As previously shown with MPTP and MPP+, the cytotoxicity of MPDP+ is dose-dependent and is consistently preceeded by complete depletion of intracellular ATP. Similar to MPP+ but not MPTP, MPDP+ causes a comparable rate and extent of cytotoxicity and ATP loss in hepatocytes pretreated with the monoamine oxidase inhibitor pargyline. Pargyline blocks hepatocyte biotransformation of MPTP to MPP+, but it has no significant effect on MPP+ accumulation after exposure to either MPDP+ or MPP+. It is concluded that MPTP is toxic to hepatocytes via its monoamine oxidase-dependent metabolism and that MPP+ is likely to be the ultimate toxic metabolite which accumulates in the cell, causing ATP depletion and eventual cell death.     

Comparative toxicity and antioxidant activity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and its monoamine oxidase B-generated metabolites in isolated hepatocytes and liver microsomes

MPTP (1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is converted by monoamine oxidase B to its putative toxic metabolite MPP+ (1-methyl-4-phenylpyridinium ion) via MPDP+ (1-methyl-4-phenyl-2,3-dihydropyridinium ion). Both the parent compound and these two major metabolites were toxic to isolated rat hepatocytes with MPDP+ being the most toxic and MPP+ the least effective. MPP+ produced a slight increase in lipid peroxidation above control levels in hepatocytes, while both MPTP and MPDP+ showed antioxidant effects. The latter two compounds also protected against chemically and nonchemically induced lipid peroxidation in rat liver microsomes. MPDP+ was effective at much lower concentrations than MPTP. MPDP+ was also markedly more efficient when NADPH was used to induce microsomal lipid peroxidation. Lipid peroxidation as a consequence of oxygen radical generation is therefore unlikely to be involved in MPTP toxicity in vitro and the rationale of using chain-breaking antioxidants as protective agents in vivo needs a more careful evaluation.     

1-Methyl-4-phenyl-2,3-dihydropyridinium is transformed by ubiquinone to the selective nigrostriatal toxin 1-methyl-4-phenylpyridinium

We have studied the interaction of coenzyme Q with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its metabolites, 1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP(+)) and 1-methyl-4-phenylpyridinium (MPP(+)), the real neurotoxin to cause Parkinson's disease. Incubation of MPTP or MPDP(+) with rat brain synaptosomes induced complete reduction of endogenous ubiquinone-9 and ubiquinone-10 to corresponding ubiquinols. The reduction occurred in a time- and MPTP/MPDP(+) concentration-dependent manner. The reduction of ubiquinone induced by MPDP(+) went much faster than that by MPTP. MPTP did not reduce liposome-trapped ubiquinone-10, but MPDP(+) did. The real toxin MPP(+) did not reduce ubiquinone in either of the systems. The reduction by MPTP but not MPDP(+) was completely prevented by pargyline, a type B monoamine oxidase (MAO-B) inhibitor, in the synaptosomes. The results indicate that involvement of MAO-B is critical for the reduction of ubiquinone by MPTP but that MPDP(+) is a reductant of ubiquinone per se. It is suggested that ubiquinone could be an electron acceptor from MPDP(+) and promote the conversion from MPDP(+) to MPP(+) in vivo, thus accelerating the neurotoxicity of MPTP.     

Neurotoxicity studies with the monoamine oxidase B substrate 1-methyl-3-phenyl-3-pyrroline

The neurotoxic properties of the parkinsonian inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) are dependent on its metabolic activation in a reaction catalyzed by centrally located monoamine oxidase B (MAO-B). This reaction ultimately leads to the permanently charged 1-methyl-4-phenylpyridinium species MPP(+), a 4-electron oxidation product of MPTP and a potent mitochondrial toxin. The corresponding 5-membered analogue, 1-methyl-3-phenyl-3-pyrroline, is also a selective MAO-B substrate. Unlike MPTP, the MAO-B-catalyzed oxidation of 1-methyl-3-phenyl-3-pyrroline is a 2-electron process that leads to the neutral 1-methyl-3-phenylpyrrole. MPP(+) is thought to exert its toxic effects only after accumulating in the mitochondria, a process driven by the transmembrane electrochemical gradient. Since this energy-dependent accumulation of MPP(+) relies upon its permanent charge, 1-methyl-3-phenyl-3-pyrrolines and their pyrrolyl oxidation products should not be neurotoxic. We have tested this hypothesis by examining the neurotoxic potential of 1-methyl-3-phenyl-3-pyrroline and 1-methyl-3-(4-chlorophenyl)-3-pyrroline in the C57BL/6 mouse model. These pyrrolines did not deplete striatal dopamine while analogous treatment with MPTP resulted in 65-73% depletion. Kinetic studies revealed that both 1-methyl-3-phenyl-3-pyrroline and its pyrrolyl oxidation product were present in the brain in relatively high concentrations. Unlike MPP(+), however, 1-methyl-3-phenylpyrrole was cleared from the brain quickly. These results suggest that the brain MAO-B-catalyzed oxidation of xenobiotic amines is not, in itself, sufficient to account for the neurodegenerative properties of a compound like MPTP. The rapid clearance of 1-methyl-3-phenylpyrroles from the brain may contribute to their lack of neurotoxicity.     

Interactions of the neurotoxic amine 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine with monoamine oxidases.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a thermal breakdown product of a meperidine-like narcotic used by drug abusers as a heroin substitute, produces Parkinsonian symptoms in humans and primates. The nigrostriatal toxicity is not due to MPTP itself but to one or more oxidation products resulting from the action of monoamine oxidase (MAO) on this tertiary allylamine. Both MAO A and B catalyse the oxidation of MPTP to the 1-methyl-4-phenyl-2,3-dihydropyridinium species (MPDP+), which undergoes further oxidation to the fully aromatic 1-methyl-4-phenylpyridinium species (MPP+). These bio-oxidations are blocked by selective inhibitors of MAO A and B. Additionally, MPTP, MPDP+ and MPP+ are competitive inhibitors of MAO A and B. The A form of the enzyme is particularly sensitive to this type of reversible inhibition. Both MAO A and B also are irreversibly inactivated by MPTP and MPDP+, but not by MPP+. This inactivation obeys the characteristics of a mechanism-based or 'suicide' process. The inactivation, which is accompanied by the incorporation of radioactivity from methyl-labelled MPTP, is likely to result from covalent modification of the enzyme.     

Reversible inhibition and mechanism-based irreversible inactivation of monoamine oxidases by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

It has been suggested (Chiba et al., Biochem. Biophys. Res. Communs. (1984) 120, 574) that the neurotoxic effects of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), which causes Parkinsonian symptoms in humans and other primates, are due to compounds resulting from the oxidation of MPTP by monoamine oxidase B in the brain. We reported recently that both monoamine oxidase A and B oxidize MPTP to MPDP+, the 2,3-dihydropyridinium form and that the reaction is accompanied by time-dependent, irreversible inactivation of the enzymes. Of the two forms of monoamine oxidase, the B enzyme oxidizes MPTP more rapidly and is also more sensitive to inactivation. We now wish to report that MPTP, as well as its oxidation products, MPDP+ and MPP+, the 4-phenylpyridinium form, are also potent reversible, competitive inhibitors of both monoamine oxidase A and B, particularly the former, and that the order of inhibition for the A enzyme is MPDP+ greater than MPP+ greater than MPTP, while for the B enzyme MPTP greater than MPDP+ greater than MPP+. We further report on the spectral changes and isotope incorporation accompanying the irreversible inactivation.     

Inhibition of NADH-linked oxidation in brain mitochondria by 1-methyl-4-phenyl-pyridine, a metabolite of the neurotoxin, 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine

1-methyl-4-phenylpyridine (MPP+), a major metabolite of the neurotoxin, 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) inhibited the ADP-stimulated and uncoupled oxidation of NADH-linked substrates by brain mitochondrial preparations. MPTP itself was ineffective. The apparent Ki's for MPP+ inhibition of pyruvate or glutamate oxidation by purified rat brain mitochondria were approximately 300 and 400 microM, respectively; with mouse brain mitochondria the values were lower, 60 and 150 microM, respectively. Succinate oxidation was unaffected by either compound. Compromise of mitochondrial oxidative capacity by MPP+ could be an important factor in mechanisms underlying the toxicity of MPTP.     

Effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its toxic metabolites on the physicochemical property of the liposomal membrane in relation to their cytochrome P-450 inhibition.

The effects of the neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and its toxic metabolites MPDP+ (1-methyl-4-phenyl-2,3-dihydropyridinium) and MPP+ (1-methyl-4-phenylpyridinium) on liposomal membrane were assessed using fluorescence-polarization and carboxyfluorescein leakage studies as well as in biological membrane preparations. Of the three compounds, MPTP was found to cause the greatest perturbation of membrane followed by MPDP+ and then MPP+. The ability of the three toxins to inhibit cytochrome P-450 enzyme activity (a microsomal membrane-bound enzyme system) was also studied and their relative potency was again found to be MPTP > MPDP+ > MPP+. The changes in the physicochemical property of the liposomal membrane can be related to the ability of the neurotoxin's ability to inhibit cytochrome P-450 activity.     

A catalyst function for MPTP in superoxide formation

We demonstrate that 1-methyl-4-phenyl-1,2-dihydropyridine (MPDP) can be generated, in an alternate pathway, from the catalyst action of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) upon the iron redox equilibrium reaction. Superoxide and ferric iron are instantaneously produced after addition of MPTP to a solution of ferrous iron. This reaction is oxygen and pH dependent. Superoxide, through a iron dependent Haber-Weiss reaction with peroxide, can generate the cytotoxic hydroxyl radical. A small portion of the superoxide reacts with MPTP to produce the reactive species X. which, in the presence of Fe+3 can also generate MPDP.     

Effects of 1-Methyl-4-Phenyl-1,2,5,6-Tetrahydropyridine and Its Metabolite 1-Methyl-4-Phenylpyridine on Acetylcholine Synthesis in Synaptosomes from Rat Forebrain

1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) and its metabolite, 1-methyl-4-phenylpyridine (MPP+), have been shown to cause a number of lesions in dopaminergic pathways of the nigro-striatal region of the brain. However, data on the effects of these neurotoxins on other aspects of brain metabolism are scarce. The data presented here show that MPTP and MPP+ inhibit glucose oxidation via the tricarboxylic acid cycle, and acetylcholine synthesis in synaptosomal preparations from rat forebrain. Monoamine oxidase B inhibitors (e.g., pargyline, MDL 72145) relieve the inhibition caused by MPTP but not MPP+. The inhibitory effects of MPP+ on glucose oxidation and acetylcholine synthesis are a consequence of the decreased glucose metabolism in synaptosomes and are consistent with its role as an inhibitor of the Complex I (NADH-CoQ reductase) of the mitochondrial respiratory chain.     

MPTP, MPP+ and mitochondrial function

1-Methyl-4-phenylpyridinium (MPP+), the putative toxic metabolite of the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), inhibited NAD(H)-linked mitochondrial oxidation at the level of Complex I of the electron transport system. MPTP and MPP+ inhibited aerobic glycolysis in mouse striatal slices, as measured by increased lactate production; MPTP-induced effects were prevented by inhibition of monoamine oxidase B activity. Several neurotoxic analogs of MPTP also form pyridinium metabolites via MAO; these MPP+ analogs were all inhibitors of NAD(H)-linked oxidation by isolated mitochondria. 2'-Methyl-MPTP, a more potent neurotoxin in mice than MPTP, was also more potent than MPTP in inducing lactate accumulation in mouse brain striatal slices. Overall, the studies support the hypothesis that compromise of mitochondrial oxidative capacity is an important factor in the mechanisms underlying the toxicity of MPTP and similar compounds.     

Impaired mutagenic activities of MPDP(+) (1-methyl-4-phenyl-2,3-dihydropyridinium) and MPP(+) (1-methyl-4-phenylpyridinium) due to their interactions with methylxanthines

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is a neurotoxin causing symptoms that resemble those observed in patients suffering from Parkinson's disease. However, in animal or human organisms, MPTP is converted to MPDP(+) (1-methyl-4-phenyl-2,3-dihydropyridinium) and further to MPP(+) (1-methyl-4-phenylpyridinium); the latter compound is the actual neurotoxin. In this report, we demonstrate that MPDP(+) and MPP(+) can form stacking complexes with methylxanthines (caffeine and penthoxifylline), which leads to significant impairment of the biological activity of these toxins (as measured by their mutagenicity).     

Effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), cyperquat (MPP+) and paraquat on isolated mitochondria from rat striatum, cortex and liver

The effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), its metabolite 1-methyl-4-phenyl pyridinium ion (MPP+, cyperquat) and a structurally-related compound paraquat on mitochondrial functions were investigated in isolated organelles from rat striatum, cortex and liver. MPTP (0.1-1.0 mM) had no significant effect on various parameters of mitochondrial oxidative phosphorylation. In contrast, MPP+ (0.5 mM) inhibited the oxidation of the nicotinamide adenine dinucleotide (NAD+)-linked substrates pyruvate and malate but not that of the flavin adenine dinucleotide (FAD+)-linked substrate succinate. Paraquat (5.0 mM) significantly stimulated basal oxygen consumption (state 4) without influencing the oxygen utilization (state 3) associated with adenosine diphosphate (ADP) phosphorylation. Thus, these structurally-related compounds have different effects on mitochondrial oxidative phosphorylation, but the organelles from striatum, cortex and liver were affected in a similar manner by these compounds.     

Relationships between the mitochondrial transmembrane potential, ATP concentration, and cytotoxicity in isolated rat hepatocytes

The relationships between mitochondrial transmembrane potential, ATP concentration, and cytotoxicity were evaluated after exposure of isolated rat hepatocytes to different mitochondrial poisons. Both the neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its fully oxidized metabolite, the 1-methyl-4-phenylpyridinium (MPP+) ion, caused a concentration- and time-dependent depolarization of mitochondrial membranes which followed ATP depletion and preceded cytotoxicity. The effect of MPTP, but not that of MPP+, was prevented by deprenyl, an inhibitor of MPTP conversion to MPP+ via monoamine oxidase type B. Addition of fructose to the hepatocyte incubations treated with either MPTP or MPP+ counteracted the loss of mitochondrial transmembrane potential. Fructose was also effective in protecting against the mitochondrial membrane depolarization as well as ATP depletion and cytotoxicity induced by antimycin. A, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, and valinomycin. Data confirm the key role played by MPP(+)-induced mitochondrial damage in MPTP toxicity and indicate that (i) ATP produced via the glycolytic pathway can be utilized by hepatocytes to maintain mitochondrial electrochemical gradient, and (ii) a loss of mitochondrial membrane potential may occur only when supplies of ATP are depleted.     

Deuterium isotope effects for the oxidation of 1-methyl-3-phenyl-3-pyrrolinyl analogues by monoamine oxidase B

The parkinsonian inducing agent, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is a cyclic tertiary allylamine exhibiting good monoamine oxidase B (MAO-B) substrate properties. MAO-B catalyzes the ring alpha-carbon 2-electron bioactivation of MPTP to yield the 1-methyl-4-phenyl-2,3-dihydropyridinium species (MPDP(+)). The corresponding 5-membered ring MPTP analogue, 1-methyl-3-phenyl-3-pyrroline, also undergoes MAO-B-catalyzed oxidation to give the 2-electron oxidation product, 1-methyl-3-phenylpyrrole. Here we report the kinetic deuterium isotope effects on V(max) and V(max)/K(m) for the steady-state oxidation of 1-methyl-3-phenyl-3-pyrroline and 1-methyl-3-(4-fluorophenyl)-3-pyrroline by baboon liver MAO-B, using the corresponding pyrroline-2,2,4,5,5-d(5) analogues as the deuterated substrates. The apparent isotope effects for the two substrates were 4.29 and 3.98 on V(max), while the isotope effects on V(max)/K(m) were found to be 5.71 and 3.37, respectively. The values reported for the oxidation of MPTP by bovine liver MAO-B with MPTP-6,6-d(2), as deuterated substrate, are (D)(V(max))=3.55; (D)(V(max)/K(m))=8.01. We conclude that the mechanism of the MAO-B-catalyzed oxidation of pyrrolinyl substrates is similar to that of the tetrahydropyridinyl substrates and that a carbon-hydrogen bond cleavage step is, at least partially, rate determining.     

Effects of 1-Methyl-4-Phenyl- 1,2,3,6-Tetrahydropyridine and 1 -Methyl-4-Phenylpyridinium Ion on ATP Levels of Mouse Brain Synaptosomes

Mouse brain synaptosomes, essentially devoid of mitochondrial contamination, were used as a model to study the effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) on the levels of ATP of neuronal terminals. Similar to known inhibitors of ATP synthesis, both MPTP and MPP+ caused a dramatic depletion of synaptosomal ATP. This depletion was dose dependent and occurred as a relatively early biochemical event in the absence of any apparent damage to synaptosomal membranes. MPP+ was more effective than its parent compound in decreasing ATP; it induced a significant loss at concentrations (10-100 microM) similar to those it reaches in the brain in vivo. MPTP-induced ATP depletion was completely prevented by the monoamine oxidase B inhibitor deprenyl, which, on the contrary, was ineffective against MPP+. As expected in view of the heterogeneous population of nerve terminals present in our synaptosomal preparations, the catecholamine uptake blocker mazindol did not significantly affect the ATP loss caused by both compounds. Data indicate that (1) administration of MPTP may cause a depletion of ATP within neuronal terminals resulting from the generation of MPP+, and (2) exposure to the levels of MPP+ reached in vivo may cause biochemical changes that are nonselective for dopaminergic terminals.     

N-methyltetrahydro-beta-carboline analogs of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin are oxidized to neurotoxic beta-carbolinium cations by heme peroxidases

2-Methyl-1,2,3,4-tetrahydro-beta-carboline (2-Me-THbetaC) and 2,9-dimethyl-1,2,3,4-tetrahydro-beta-carboline (2,9-diMe-THbetaC) are naturally occurring analogs of the Parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), whereas their corresponding aromatic 2-methyl-beta-carbolinium cations resemble 1-methyl-4-phenylpyridinium (MPP(+)) and are considered potential toxins involved in Parkinson's disease (PD). To become toxicants, 2-methyltetrahydro-beta-carbolines need to be oxidized (aromatized) by human metabolic enzymes to pyridinium-like (beta-carbolinium) cations as occur with MPTP/MPP(+) model. In contrast to MPTP, human MAO-A or -B were not able to oxidize 2-Me-THbetaC to pyridinium-like cations. Neither, cytochrome P-450 2D6 or a mixture of six P450 enzymes carried out this oxidation in a significant manner. However, 2-Me-THbetaC and 2,9-diMe-THbetaC were efficiently oxidized by horseradish peroxidase (HRP), lactoperoxidase (LPO), and myeloperoxidase (MPO) to 2-methyl-3,4-dihydro-beta-carbolinium cations (2-Me-DHbetaC(+), 2,9-diMe-DHbetaC(+)) as the main products, and detectable amount of 2-methyl-beta-carbolinium cations (2-Me-betaC(+), 2,9-diMe-betaC(+)). The apparent kinetic parameters (k(cat), k(4)) were similar for HRP and LPO and higher for MPO. Peroxidase inhibitors (hydroxylamine, sodium azide, and ascorbic acid) highly reduced or abolished this oxidation. Although MPTP was not oxidized by peroxidases; its intermediate metabolite 1-methyl-4-phenyl-2,3-dihydropyridinium cation (MPDP(+)) was efficiently oxidized to MPP(+) by heme peroxidases. It is concluded that heme peroxidases could be key catalysts responsible for the aromatization (bioactivation) of endogenous and naturally occurring N-methyltetrahydro-beta-carbolines and related protoxins to toxic pyridinium-like cations resembling MPP(+), suggesting a role for these enzymes in toxicological and neurotoxicological processes.     

EPR kinetic studies of superoxide radicals generated during the autoxidation of 1-methyl-4-phenyl-2,3-dihydropyridinium, a bioactivated intermediate of parkinsonian-inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

1-Methyl-4-phenyl-2,3-dihydropyridinium (MPDP+), a metabolic product of the nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), has been shown to generate superoxide radicals during its autoxidation process. The generation of superoxide radicals was detected as a 5,5-dimethyl-1-pyrroline-N-oxide (DMPO).O2- spin adduct by spin trapping in combination with EPR techniques. The rate of formation of spin adduct was dependent not only on the concentrations of MPDP+ and oxygen but also on the pH of the system. Superoxide dismutase inhibited the spin adduct formation in a dose-dependent manner. The ability of DMPO to trap superoxide radicals, generated during the autoxidation of MPDP+, and of superoxide dismutase to effectively compete with this reaction for the available O2-, has been used as a convenient competition reaction to quantitatively determine various kinetic parameters. Thus, using this technique the rate constant for scavenging of superoxide radical by superoxide dismutase was found to be 7.56 x 10(9) M-1 s-1. The maximum rate of superoxide generation at a fixed spin trap concentration using different amounts of MPDP+ was found to be 4.48 x 10(-10) M s-1. The rate constant (K1) for MPDP+ making superoxide radical was found to be 3.97 x 10(-6) s-1. The secondary order rate constant (KDMPO) for DMPO-trapping superoxide radicals was found to be 10.2 M-1 s-1. The lifetime of superoxide radical at pH 10.0 was calculated to be 1.25 s. These values are in close agreement to the published values obtained using different experimental techniques. These results indicate that superoxide radicals are produced during spontaneous oxidation of MPDP+ and that EPR spin trapping can be used to determine the rate constants and lifetime of free radicals generated in aqueous solutions. It appears likely that the nigrostriatal toxicity of MPTP/MPDP+ leading to Parkinson's disease may largely be due to the reactivity of these radicals.     

Studies on the Neurotoxicity of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine: Inhibition of NAD-Linked Substrate Oxidation by Its Metabolite, 1-Methyl-4-Phenylpyridinium

Abstract: The effects of the parkinsonism-inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its 4-electron oxidation product 1-methyl-4-phenylpyridinium (MPP⁺) were studied in isolated mitochondria and in mouse brain striatal slices. ADP-stimulated oxidation of NAD-linked substrates was inhibited in a time-dependent manner by MPP⁺ (0.1–0.5 m M ), but not MPTP, in mitochondria prepared from rat brain, mouse brain, or rat liver. Under identical conditions, succinate oxidation was relatively unaffected. In neostriatal slices prepared from the mouse, a species susceptible to the dopaminergic neurotoxicity of MPTP, incubation with either MPP⁺ or MPTP caused metabolic changes consistent with inhibition of mitochondnial oxidation, i.e., an increase in the formation of lactate and accumulation of the amino acids glutamate and alanine with concomitant decreases in glutamine and aspartate levels. The changes resulting from incubation with MPTP were prevented by the monoamine oxidase inhibitor pargyline, which blocks formation of MPP⁺ from MPTP. The results suggest that compromise of mitochondrial function and its metabolic sequelae within dopaminergic neurons could be an important factor in the neurotoxicity observed after MPTP administration.