Structural insights into vesicular monoamine storage and drug interactions.

Biogenic monoamines-vital transmitters orchestrating neurological, endocrinal and immunological functions1-5-are stored in secretory vesicles by vesicular monoamine transporters (VMATs) for controlled quantal release6,7. Harnessing proton antiport, VMATs enrich monoamines around 10,000-fold and sequester neurotoxicants to protect neurons8-10. VMATs are targeted by an arsenal of therapeutic drugs and imaging agents to treat and monitor neurodegenerative disorders, hypertension and drug addiction1,8,11-16. However, the structural mechanisms underlying these actions remain unclear. Here we report eight cryo-electron microscopy structures of human VMAT1 in unbound form and in complex with four monoamines (dopamine, noradrenaline, serotonin and histamine), the Parkinsonism-inducing MPP+, the psychostimulant amphetamine and the antihypertensive drug reserpine. Reserpine binding captures a cytoplasmic-open conformation, whereas the other structures show a lumenal-open conformation stabilized by extensive gating interactions. The favoured transition to this lumenal-open state contributes to monoamine accumulation, while protonation facilitates the cytoplasmic-open transition and concurrently prevents monoamine binding to avoid unintended depletion. Monoamines and neurotoxicants share a binding pocket that possesses polar sites for specificity and a wrist-and-fist shape for versatility. Variations in this pocket explain substrate preferences across the SLC18 family. Overall, these structural insights and supporting functional studies elucidate the mechanism of vesicular monoamine transport and provide the basis to develop therapeutics for neurodegenerative diseases and substance abuse.

Caenorhabditis elegans Model Studies Show MPP+ Is a Simple Member of a Large Group of Related Potent Dopaminergic Toxins.

Although the causes of Parkinson's disease (PD) are not fully understood, the consensus is that a combination of genetic and environmental factors plays a major role. The discovery that the synthetic chemical, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-derived N-methyl-4-phenylpyridinium (MPP+), recapitulates major pathophysiological characteristics of PD in humans and other mammals has provided the strongest support for this possibility; however, several key aspects of the mechanism remain unclear. In contrast to the widely accepted view that MPP+ is structurally unique and optimal for selective dopaminergic toxicity, previous in vitro studies have suggested that MPP+ is most likely a simple member of a large group of related dopaminergic toxins. Here we provide first in vivo evidence to support the above possibility using Caenorhabditis elegans PD models. We also provide in vivo evidence to show that the inherent predisposition of dopaminergic neurons to produce high oxidative stress and related downstream effects when exposed to MPP+ and related mitochondrial toxins is responsible for their selective vulnerability to these toxins. More significantly, present findings suggest that if this broad group of MPP+ related dopaminergic toxins are present in work places or in the environment, they could cause far-reaching public health consequences.

Incorporation of a Biguanide Scaffold Enhances Drug Uptake by Organic Cation Transporters 1 and 2.

Membrane transporters play a significant role in the transport of many endogenous and exogenous compounds. The knowledge of transporter substrate requirements has allowed further development of drugs that utilize them to ensure tissue permeation. In this study, we demonstrate that inclusion of a biguanide functionality can potentiate uptake by the organic cation transporters 1 and 2 (OCT1 and OCT2). We synthesized 18 pairs of structurally diverse compounds, each pair consisting of a parent amino compound and its biguanide analog; and then assessed their cellular uptake in HEK293 cells overexpressing human OCT1 or OCT2. Our results show that addition of the biguanide significantly improved OCT1- and OCT2-mediated transport for the majority of compounds. The biguanides also inhibited the uptake of prototypical substrates of both transporters, 1-methyl-4-phenylpyridinium (MPP+) and metformin. We found that molecular weight, molecular volume, Log D (pH 7.4), and accessible surface area were important determinants of OCT2 substrates, but none of these parameters was a significant factor for OCT1. More so, the inhibition of MPP+ uptake correlated linearly with that of metformin uptake for the tested biguanides in both cell lines. Taken together, we conclude that the inclusion of the biguanide scaffold in nonsubstrates of OCT1 and OCT2 increase their propensity to become substrates and inhibitors for these transporters.

Retina-to-Blood Transport of 1-Methyl-4-Phenylpyridinium Involves Carrier-Mediated Process at the Blood-Retinal Barrier.

1-Methyl-4-phenylpyridinium (MPP+) transport at the blood-retinal barrier (BRB) was investigated. The retinal uptake index estimated for [3H]MPP+ was similar to that of [3H]d-mannitol, and was insensitive to unlabeled MPP+, suggesting no positive evidence to support the involvement of carrier-mediated transport in the blood-to-retina transport of MPP+ at the BRB. A microdialysis investigation showed that the concentration of [3H]MPP+ in the vitreous humor decreased in a biexponential manner, and the rate constant for [3H]MPP+ elimination during the terminal phase was greater than that of [14C]d-mannitol. The inhibition study of [3H]MPP+ elimination showed its substrate specificity, suggesting that the retina-to-blood transport of MPP+ at the BRB involves a carrier-mediated process. The in vitro study with model cells showed the concentration-dependent transport of MPP+, supporting carrier-mediated MPP+ transport at the inner and outer BRB, and suggested membrane potential-sensitive and Na+-, Cl--, and pH-insensitive MPP+ transport at the BRB. In the in vitro inhibition study, the transport of [3H]MPP+ was significantly inhibited by organic cations, and further reverse transcription PCR analysis and knockdown study suggested that the retina-to-blood transport of MPP+ at the BRB is carried out by an unknown transporter of which transport function is similar to plasma membrane monoamine transporter (PMAT/SLC29A4).

Mitochondrial pyruvate carrier regulates autophagy, inflammation, and neurodegeneration in experimental models of Parkinson's disease.

Mitochondrial and autophagic dysfunction as well as neuroinflammation are involved in the pathophysiology of Parkinson's disease (PD). We hypothesized that targeting the mitochondrial pyruvate carrier (MPC), a key controller of cellular metabolism that influences mTOR (mammalian target of rapamycin) activation, might attenuate neurodegeneration of nigral dopaminergic neurons in animal models of PD. To test this, we used MSDC-0160, a compound that specifically targets MPC, to reduce its activity. MSDC-0160 protected against 1-methyl-4-phenylpyridinium (MPP+) insult in murine and cultured human midbrain dopamine neurons and in an α-synuclein-based Caenorhabditis elegans model. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, MSDC-0160 improved locomotor behavior, increased survival of nigral dopaminergic neurons, boosted striatal dopamine levels, and reduced neuroinflammation. Long-term targeting of MPC preserved motor function, rescued the nigrostriatal pathway, and reduced neuroinflammation in the slowly progressive Engrailed1 (En1+/-) genetic mouse model of PD. Targeting MPC in multiple models resulted in modulation of mitochondrial function and mTOR signaling, with normalization of autophagy and a reduction in glial cell activation. Our work demonstrates that changes in metabolic signaling resulting from targeting MPC were neuroprotective and anti-inflammatory in several PD models, suggesting that MPC may be a useful therapeutic target in PD.

General Scheme for Oxidative Quenching of a Copper Bis-Phenanthroline Photosensitizer for Light-Driven Hydrogen Production.

A new, general reaction scheme for photocatalytic hydrogen production is presented based on oxidative quenching of a homoleptic copper(I) bis-1,10-phenanthroline photosensitizer (PS) by 1-methyl-4-phenyl-pyridinium (MPP(+) ) as the electron relay and subsequent regeneration of the so formed copper(II) complex by a sacrificial electron donor. Electron transfer from the relay to various cobalt based water reduction catalysts and subsequent H2 production was shown to close the catalytic cycle. Transient absorption experiments unambiguously confirmed the proposed pathway, both the oxidative quenching and subsequent regeneration of oxidized PS. Photocatalytic test runs further confirmed the role of MPP(+) and up to 10 turnovers were achieved in the relay. The performance limiting factor of the system was shown to be the decomplexation of the copper PS. Quantum yields of the system were 0.03 for H2 production, but 0.6 for MPP(.) formation, clearly indicating that unproductive pathways still prevail.

MicroRNA-7 Promotes Glycolysis to Protect against 1-Methyl-4-phenylpyridinium-induced Cell Death.

Parkinson disease is associated with decreased activity of the mitochondrial electron transport chain. This defect can be recapitulated in vitro by challenging dopaminergic cells with 1-methyl-4-phenylpyridinium (MPP(+)), a neurotoxin that inhibits complex I of electron transport chain. Consequently, oxidative phosphorylation is blocked, and cells become dependent on glycolysis for ATP production. Therefore, increasing the rate of glycolysis might help cells to produce more ATP to meet their energy demands. In the present study, we show that microRNA-7, a non-coding RNA that protects dopaminergic neuronal cells against MPP(+)-induced cell death, promotes glycolysis in dopaminergic SH-SY5Y and differentiated human neural progenitor ReNcell VM cells, as evidenced by increased ATP production, glucose consumption, and lactic acid production. Through a series of experiments, we demonstrate that targeted repression of RelA by microRNA-7, as well as subsequent increase in the neuronal glucose transporter 3 (Glut3), underlies this glycolysis-promoting effect. Consistently, silencing Glut3 expression diminishes the protective effect of microRNA-7 against MPP(+). Further, microRNA-7 fails to prevent MPP(+)-induced cell death when SH-SY5Y cells are cultured in a low glucose medium, as well as when differentiated ReNcell VM cells or primary mouse neurons are treated with the hexokinase inhibitor, 2-deoxy-d-glucose, indicating that a functional glycolytic pathway is required for this protective effect. In conclusion, microRNA-7, by down-regulating RelA, augments Glut3 expression, promotes glycolysis, and subsequently prevents MPP(+)-induced cell death. This protective effect of microRNA-7 could be exploited to correct the defects in oxidative phosphorylation in Parkinson disease.

Mutational analysis of the high-affinity zinc binding site validates a refined human dopamine transporter homology model.

The high-resolution crystal structure of the leucine transporter (LeuT) is frequently used as a template for homology models of the dopamine transporter (DAT). Although similar in structure, DAT differs considerably from LeuT in a number of ways: (i) when compared to LeuT, DAT has very long intracellular amino and carboxyl termini; (ii) LeuT and DAT share a rather low overall sequence identity (22%) and (iii) the extracellular loop 2 (EL2) of DAT is substantially longer than that of LeuT. Extracellular zinc binds to DAT and restricts the transporter's movement through the conformational cycle, thereby resulting in a decrease in substrate uptake. Residue H293 in EL2 praticipates in zinc binding and must be modelled correctly to allow for a full understanding of its effects. We exploited the high-affinity zinc binding site endogenously present in DAT to create a model of the complete transmemberane domain of DAT. The zinc binding site provided a DAT-specific molecular ruler for calibration of the model. Our DAT model places EL2 at the transporter lipid interface in the vicinity of the zinc binding site. Based on the model, D206 was predicted to represent a fourth co-ordinating residue, in addition to the three previously described zinc binding residues H193, H375 and E396. This prediction was confirmed by mutagenesis: substitution of D206 by lysine and cysteine affected the inhibitory potency of zinc and the maximum inhibition exerted by zinc, respectively. Conversely, the structural changes observed in the model allowed for rationalizing the zinc-dependent regulation of DAT: upon binding, zinc stabilizes the outward-facing state, because its first coordination shell can only be completed in this conformation. Thus, the model provides a validated solution to the long extracellular loop and may be useful to address other aspects of the transport cycle.

Evaluation of the oxidation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to toxic pyridinium cations by monoamine oxidase (MAO) enzymes and its use to search for new MAO inhibitors and protective agents.

Monoamine oxidase (MAO) enzymes catalyze the oxidative deamination of amines and neurotransmitters and inhibitors of MAO are useful as neuroprotectants. This work evaluates the human MAO-catalyzed oxidation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a dopaminergic neurotoxin, to the directly-acting neurotoxic metabolites, 1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP(+)) and 1-methyl-4-phenylpyridinium (MPP(+)) measured by High-Performance Liquid Chromatography (HPLC), and this approach is subsequently used as a new method for screening of MAO inhibitors and protective agents. Oxidation of MPTP by human MAO-B was more efficient than by MAO-A. R-Deprenyl, a known neuroprotectant, norharman (ß-carboline), 5-nitroindazole and menadione (vitamin K3) inhibited MAO-B and reduced the formation of toxic pyridinium cations. Clorgyline and the ß-carbolines, harman and norharman, inhibited the oxidation of MPTP by MAO-A. Cigarette smoke, as well as the naturally occurring ß-carbolines (norharman and harman) isolated from smoke and coffee inhibited the oxidation of MPTP by MAO-B and/or MAO-A, suggesting protective effects against MPTP. The results show the suitability of the approach used to search for new MAO inhibitors with eventual neuroprotective activity.

Liquid chromatographic-electrospray mass spectrometric determination of 1-methyl-4-phenylpyridine (MPP+) in discrete regions of murine brain.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is widely used as a neurotoxin in several models of Parkinson's disease in mice. MPTP is metabolized to 1-methyl-4-phenylpyridinium (MPP(+)), which is a mitochondrial toxicant of central dopamine (DA) neurons. There are species, strain, and age differences in sensitivity to MPTP. Simultaneous measurement of the MPTP active metabolite MPP(+) and dopamine (DA) in the brain would be helpful in mechanistic studies of this neurotoxin. The objective of this study was to develop a liquid chromatography-mass spectrometry (LC/MS) method for analysis of MPTP and MPP(+) in brain tissue and correlate these in the same sample with changes in DA measured via HPLC coupled with electrochemical detection. Twenty-five C57BL/6J7 8-week old female mice were used in the study. Mice were given a single subcutaneous injection of MPTP (20 mg/kg) and were sacrificed 1, 2, 4, or 8 h later. Zero time control mice received an injection of 0.9% normal saline (10 ml/kg) and were killed 1 h later. Brains were rapidly harvested and quickly frozen, and microdissected brain regions were placed in 0.1 M phosphate-citric acid buffer containing 20% methanol (pH 2.5). A new LC/MS method was successfully developed that utilized selected reaction monitoring (SRM) of MPP(+) m/z 170→127, 170→128, and 170→154 fragmentation for quantitation and area ratios (m/z 127)/(m/z 128) and (m/z 154)/(128) for identity confirmation. A similar SRM strategy from m/z 174 was unable to detect any significant levels of MPTP down to 0.4 ppb. According to this method, MPP(+) was detected in the nucleus accumbens (NA) and the striatum (ST), with the levels in the NA being 3-times higher than those in the ST. The advantage of this approach is that the tissue buffer used in this procedure allowed concurrent measurement of striatal DA, thus enabling direct correlation between accumulation of tissue MPP(+) and depletion of DA concentrations in discrete regions of the brain.

Stereoselective binding of chiral ligands to single nucleotide polymorphisms of the human organic cation transporter-1 determined using cellular membrane affinity chromatography.

Membranes from stably transfected cell lines that express two point mutations of the human organic cation transporter-1 (hOCT1), R488M and G465R, have been immobilized on the immobilized artificial membrane (IAM) liquid chromatographic stationary phase to form two cellular membrane affinity chromatography (CMAC) columns, CMAC(hOCT1(G465R)) and CMAC(hOCT1(R488M)). Columns were created using both stationary phases, and frontal displacement chromatography experiments were conducted using [(3)H] MMP(+) (1-methyl-4-phenylpyridinium) as the marker ligand and various displacers, including the single enantiomers of verapamil, fenoterol, and isoproterenol. The chromatographic data obtained were used to refine a previously developed pharmacophore for hOCT1.

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.

Distinct effects of atypical 1,4-dihydropyridines on 1-methyl-4-phenylpyridinium-induced toxicity.

Our previous data obtained from in vivo experiments demonstrated high neuroprotective effects of three novel atypical neuronal non-calcium antagonistic 1,4-dihydropyridine (DHP) derivatives cerebrocrast, glutapyrone and tauropyrone. The present studies were carried out in vitro to clarify, at least in part, their mechanism of action in primary culture of cerebellar granule cells by use of 1-methyl-4-phenylpyridinium (MPP+) as a neurotoxic agent which causes dramatic oxidative stress. Cerebrocrast (highly lipophilic, with a classical two-ring structure) dose-dependently (0.01-10.0 microM, EC50 = 13 nM) reduced MPP+-induced cell death. At the same time, the calcium antagonist nimodipine (reference drug) protected cell death at much higher concentrations (EC50 = 12.4 microM). Cerebrocrast decreased also the generation of reactive oxygen species and loss of mitochondrial membrane potential. In contrast, low lipophilic amino acid-containing DHPs glutapyrone and tauropyrone (glutamate- and taurine-containing, correspondingly) were without significant effects indicating their distinct mode of action in comparison to cerebrocrast. We have demonstrated for the first time an ability of atypical non-calcium antagonistic DHP cerebrocrast (which has classical DHP structure elements and high lipophilicity) to protect MPP+-induced deterioration of mitochondrial bioenergetics. One may suggest mitochondria as an essential intracellular target for the neuroprotective action of cerebrocrast and indicate its usefulness in the treatment of Parkinson's disease.

2,9-Dimethyl-beta-carbolinium, a neurotoxin occurring in human brain, is a potent inducer of apoptosis as 1-methyl-4-phenylpyridinium.

The causes of neurodegeneration are not well understood. However, the role of environmental and endogenous toxins is receiving much attention. In this study, we compared the synthetic neurotoxin 1-methyl-4-phenyl-pyridinium with beta-carbolines occurring in human brain. Methylation of both nitrogens is necessary to convert a beta-carboline into a potent inhibitor of mitochondrial complex I. The respective beta-carboline, 2,9-dimethyl-beta-carbolinium ion is neurotoxic in rats. To investigate the underlying mechanisms, we incubated mouse neuroblastoma 2A cells with 2,9-dimethyl-beta-carbolinium ion, and compared the findings with effects of norharman, the precursor beta-carboline of methylated derivatives, and with 1-methyl-4-phenyl-pyridinium. 2,9-Dimethyl-beta-carbolinium ion caused a significant increase of reactive oxygen species (higher efficiency than 1-methyl-4-phenyl-pyridinium) and of mitochondrial membrane potential within the first minutes. After 60 min, the membrane potential dissipated. Concomitantly, the levels of glutathione increased in 2,9-dimethyl-beta-carbolinium ion but not in 1-methyl-4-phenyl-pyridinium treated cells. After 24 h effector caspases 3 and 7 were activated and the number of apoptotic cells increased as revealed by fluorescence-activated cell sorting cytometry. When incubated longer (48 h), cells underwent late apoptosis/secondary necrosis as shown by fluorescence-activated cell sorting analysis and confirmed qualitatively by an electron microscopy study. The effects of 2,9-dimethyl-beta-carbolinium ion on apoptotic changes were similar to those induced by 1-methyl-4-phenyl-pyridinium(,) while norharman showed only a weak potency at the very high doses. To investigate whether 2,9-dimethyl-beta-carbolinium ion is neurotoxic under in vivo conditions and whether only dopaminergic neurones are affected we conducted a dose-response study. Three weeks after injection of 2,9-dimethyl-beta-carbolinium ion in the substantia nigra we found a dose-dependent decrease of dopamine and its metabolites in the striatum of rats. The levels of 5-hydroxytryptamine were diminished although the decrease was less. The levels of noradrenaline increased after some doses. The findings strongly suggest an important role of endogenous beta-carbolines in neurodegeneration with apoptosis as the predominant mechanism.

Heat shock proteins protect both MPP(+) and paraquat neurotoxicity.

The exposure of immortalized rat neuroblast cells to MPP(+) and paraquat results in cell death. Heat shock pre-treatment prior to the addition of MPP(+) and paraquat significantly reduced cell death and led to an increase in the synthesis of Hsp 27 and Hsp70 proteins. Quercetin inhibits the synthesis of heat shock proteins (Hsp) and prevents their protective effect, which suggests that this protection was dependent on the Hsps synthesis. These data indicate that heat shock protects cells from the toxic effect of MPP(+) and paraquat. These results and the structural similarity between paraquat and MPP(+) support the role of paraquat as a putative risk factor in the etiology of Parkinson's disease.

A conserved glutamate residue in transmembrane helix 10 influences substrate specificity of rabbit OCT2 (SLC22A2).

OCT1 and OCT2 are involved in renal secretion of cationic drugs. Although they have similar selectivity for some substrates (e.g. tetraethylammonium (TEA)), they have distinct selectivities for others (e.g. cimetidine). We postulated that "homolog-specific residues," i.e. the 24 residues that are conserved in OCT1 orthologs as one amino acid and in OCT2 as a different one, influence homolog-specific selectivity and examined the influence on substrate binding of three of these conserved residues that are found in the C-terminal half of the rabbit orthologs of OCT1/2. The N353L and R403I substitutions (OCT2 to OCT1) did not significantly change the properties of OCT2. However, the E447Q replacement shifted substrate selectivity toward an OCT1-like phenotype. Substitution of glutamate with cationic amino acids (E447K and E447R) abolished transport activity, and the E447L mutant displayed markedly reduced transport of TEA and cimetidine while retaining transport of 1-methyl-4-phenylpyridinium. In a novel homology model of the three-dimensional structure of OCT2, Glu(447) was found in a putative docking region within a hydrophilic cleft of the protein. In addition, six residues identified in separate studies as exerting significant effects on OCT binding were also found within the putative cleft region. There was a significant correlation (r(2) = 0.82) between the IC(50) values for inhibition of TEA transport by 14 different compounds and their calculated K(D) values for binding to the model of rabbit OCT2. The results suggest that homology modeling offers an opportunity to direct future site-directed studies of OCT/substrate interaction.

Induction of phenotypes resembling CuZn-superoxide dismutase deletion in wild-type yeast cells: an in vivo assay for the role of superoxide in the toxicity of redox-cycling compounds.

Yeast (Saccharomyces cerevisiae) lacking the enzyme CuZn-superoxide dismutase (sod1delta) display a large number of dioxygen sensitive phenotypes, such as amino acid auxotrophies, sensitivity to elevated temperatures, and sensitivity to 100% dioxygen, which are attributed to superoxide stress. Such cells are exquisitely sensitive to small amounts of the herbicide paraquat (methyl viologen), which is known to produce high fluxes of superoxide in vivo via a redox-cycling mechanism. We report that dioxygen sensitive phenotypes similar to those seen in sod1delta cells can be induced in wild-type cells by treatment with moderate concentrations of paraquat or diquat, another bipyridyl herbicide, providing strong evidence that the mechanism of toxicity for both of these compounds is attributable to superoxide stress. Certain redox-cycling quinone compounds (e.g., menadione and plumbagin) are also far more toxic toward sod1delta than to wild type. However, treatment of wild-type yeast with menadione or plumbagin did not induce sod1delta-like phenotypes, although toxicity was evident. Thus, their toxicity in wild type cells is predominantly, but not exclusively, due to mechanisms unrelated to superoxide production. Further evidence for a different basis of toxicity toward wild-type yeast in these two classes of redox-cycling compounds includes the observations that (i) growth in low oxygen alleviated the effects of paraquat and diquat but not those of menadione or plumbagin and (ii) activity of the superoxide sensitive enzyme aconitase is affected by very low concentrations of paraquat but only by higher, growth inhibitory concentrations of menadione. These results provide the basis for an easy qualitative assay of the contribution of redox-cycling to the toxicity of a test compound. Using this method, we analyzed the Parkinsonism-inducing compound 1-methyl-4-phenylpyridinium and found that redox cycling and superoxide toxicity are not the predominant factor in its toxic mechanism.

Identification of an N-methyl-4-phenylpyridinium-like metabolite of the antidiarrheal agent loperamide in human liver microsomes: underlying reason(s) for the lack of neurotoxicity despite the bioactivation event.

In contrast with the Parkinson's-like effects associated with the mitochondrial neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and the neuroleptic agent haloperidol, there exist no reports on adverse central nervous system (CNS) effects with the structurally related N-substituted-4-arylpiperidin-4-ol derivative and antidiarrheal agent loperamide. Although this difference can be attributed to loperamide's P-glycoprotein substrate properties that prevent it from accessing the brain, an alternative possibility is that loperamide metabolism in humans is different from that of MPTP and haloperidol and does not involve bioactivation to a neurotoxic pyridinium species. In the current study, loperamide bioactivation was examined with particular focus on identification of pyridinium metabolites. A NADPH-dependent disappearance of loperamide was observed in both rat and human liver microsomes (human t(1/2) = 13 min; rat t(1/2) = 22 min). Loperamide metabolism was similar in human and rat and involved N-dealkylation to N-desmethylloperamide (M3) as the principal metabolic fate. Other routes of loperamide biotransformation included N- and C-hydroxylation to the loperamide-N-oxide (M4) and carbinolamide (M2) metabolites, respectively. Furthermore, the formation of an additional metabolite (M5) was also discernible in human and rat liver microsomes. The structure of M5 was assigned to the pyridinium species (LPP(+)) based on comparison of the liquid chromatography/tandem mass spectrometry characteristics to the pyridinium obtained from loperamide via a chemical reaction. Loperamide metabolism in human microsomes was sensitive to ketoconazole and bupropion treatment, suggesting P4503A4 and -2B6 involvement. Recombinant P4503A4 catalyzed all of the loperamide biotransformation pathways in human liver microsomes, whereas P4502B6 was only responsible for N-dealkylation and N-oxidation routes. The wide safety margin of loperamide (compared with MPTP and haloperidol) despite metabolism to a potentially neurotoxic pyridinium species likely stems from a combination of factors that include a therapeutic regimen normally restricted to a few days and the fact that loperamide and perhaps LPP(+) are P-glycoprotein substrates and are denied entry into the CNS. The differences in safety profile of haloperidol and loperamide despite a common bioactivation event supports the notion that not all compounds undergoing bioactivation in vitro will necessarily elicit a toxicological response in vivo.

High-performance liquid chromatography/tandem mass spectrometry assay for the determination of 1-methyl-4-phenyl pyridinium (MPP+) in brain tissue homogenates.

A high-throughput liquid chromatography/tandem mass spectrometry method has been developed for the quantitative assessment of 1-methyl-4-phenylpyridinium (MPP+) in brain tissue samples. This separation is based on reversed phase chromatography using formic acid and acetonitrile as the mobile phase. Using gradient separation conditions, MPP+ was resolved within 5 min and detected using tandem mass spectrometry in the positive ion electrospray mode. The limit of detection for MPP+ was found to be 1 fmol on column with a signal to noise ratio of 3:1. The assay has been used routinely in our laboratory for the measurement of MPP+ levels in brain tissue from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, and can be used to distinguish neuroprotective efficacy and monoamine oxidase inhibition.

MPP+: mechanism for its toxicity in cerebellar granule cells.

Cerebellar granule cells constitute the largest homogeneous neuronal population of the mammalian brain. However, they are not often used in studies that involve MPP+-neurotoxicity. Currently, it is known that the toxicity of MPP+ in cerebellar granule cells as well as in other models, including dopaminergic cells, results from activation of the apoptotic machinery after an initial oxidative burst with mitochondrial damage and energetic failure. Therefore, cerebellar granule cells serve as a good model to investigate the MPP+ effects and to study in vitro the molecular mechanism implicated in the genesis of Parkinson's disease.