Inhibition of VMAT-2 and DT-Diaphorase Induce Cell Death in a

Nov 16, 2006 - Substantia Nigra-Derived Cell LinesAn Experimental Cell Model ... substantia nigra by inhibiting VMAT-2 with reserpine to increase free...
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Chem. Res. Toxicol. 2007, 20, 776-783

Inhibition of VMAT-2 and DT-Diaphorase Induce Cell Death in a Substantia Nigra-Derived Cell LinesAn Experimental Cell Model for Dopamine Toxicity Studies Patricio Fuentes, Irmgard Paris, Melissa Nassif, Pablo Caviedes, and Juan Segura-Aguilar* Molecular and Clinical Pharmacology, ICBM, Faculty of Medicine, Casilla 70000, Santiago-7, Chile ReceiVed NoVember 16, 2006

We have induced intracellular dopamine oxidation to aminochrome in RCSN-3 cells derived from rat substantia nigra by inhibiting VMAT-2 with reserpine to increase free cytosolic dopamine concentration, to study aminochrome-dependent neurotoxicity in the absence of exogenous oxidizing agents such as metals, which may potentiate an aminochrome cytotoxic effect. The expression of VMAT-2 in RCSN-3 cells was determined by reverse transcriptase-polymerase chain reaction and immunocytochemistry. We observed double membrane bodies containing melanin when RCSN-3 cells were incubated with 100 µM dopamine by using transmission electron microscopy. No significant difference in the cell death was observed when the cells were treated 100 µM dopamine and 25 µM reserpine in the absence or presence of 100 µM dicoumarol, an inhibitor of DT-diaphorase. The lack of effect was due to the inhibitory action of 25 µM reserpine on DT-diaphorase (Ki ) 24 µM). However, a significant increase in the cell death was observed when DT-diaphorase was inhibited when the cells were incubated with 1 µM reserpine and 100 µM dopamine for 12 h since at this concentration reserpine inhibits VMAT-2 but not DTdiaphorase. Under this condition, we observed (i) the formation of blebbing; (ii) chromatin condensation accompanied by the formation of massive patches in contact with the nuclear membrane; (iii) the smoothness of the cell’s surface, that is, lack of surface microprojections; and (iv) mitochondrial damage characterized by disruption of cristae architecture, which remains closely packed; disorganization of the mitochondrial matrix due to separation of the outer membrane from the internal membrane and considerable enlargement of the intermembrane space; and disruption of the external mitochondrial membrane determined by transmission electron microscopy. These results support the proposed neuroprotective role of DT-diaphorase against aminochrome neurotoxicity, and it suggests that RCSN-3 cells incubated with reserpine and dopamine are an excellent and more physiological cellular experimental model to study the role of dopamine oxidation in neurotoxic effects of dopamine. Introduction Dopamine oxidation results in the formation of the o-quinone aminochrome (1-9), which is the precursor of neuromelanin (9). The neuromelanin is a naturally occurring pigment in the human nervous system that accumulates with age (10), especially in catecholaminergic neurons from substancia nigra and locus coeruleus (11, 12). The synthesis of neuromelanin is hypothesized to be the result of the cell’s defense mechanism against high oxidative stress (12). In the presence of O2, dopamine oxidizes spontaneously to aminochrome without the necessity of metal-ion catalysis (13). Parkinson’s disease is primarily the result of a selective loss of the neuromelanincontaining dopamine neurons of the substantia nigra while the unpigmented dopaminergic neurons are spared (14, 15). In dopaminergic neurons, intracellular dopamine is efficiently incorporated into vesicles by vesicular monoamine transporter-2 (VMAT-2) for neuronal transmission or storage. The low pH inside the vesicles prevents oxidation of the catechol structure of dopamine to the o-quinone aminochrome. Therefore, VMAT seems to play a very important role in preventing dopamine autoxidation in the cytosol by keeping dopamine at a low pH (16). In addition, monoamine oxidase (MAO) also prevents dopamine oxidation to aminochrome by degrading free dopam* To whom correspondence should be addressed. Tel: +56 2 678 6057. Fax: 56 2 737 2783. E-mail: [email protected].

ine when this enzyme catalyzes the oxidative deamination of free dopamine. However, despite the existence of VMAT and MAO, dopamine autoxidation to aminochrome and its polymerization to form neuromelanin occur in substantia nigra’s dopaminergic neurons. One possible explanation is because dopamine autoxidation to aminochrome occurs when VMAT and MAO enzymatic capacity is surpassed. Aminochrome formed from dopamine autoxidation has four possible pathways in dopaminergic neurons: (i) polymerization to neuromelanin; (ii) formation of adducts with R-synuclein stabilizing the neurotoxic protofibrils (17); (iii) one-electron reduction of aminochrome to leukoaminochrome o-semiquinone radical, which has been proposed to be one of the major sources for endogenous generation of reactive species involved in the degenerative process leading to Parkinson’s disease (8, 1822); and (iv) two-electron reduction of aminochrome, which prevents one-electron reduction of aminochrome, catalyzed by DT-diaphorase (EC.1.6.99.2). All of the flavoenzymes found in the cytosol in the glycolysis pathway or in the mitochondria (ubiquinone reductase, NADH cytochrome C reductase) are able to catalyze one-electron reduction of aminochrome with the exception of DT-diaphorase, the unique flavoenzyme (quinone reductase) that catalyzes two-electron reduction of quinones. This enzyme is localized in both dopaminergic neurons and glia cells (23) and prevents toxic effects of CuSO4, FeCl3, and aminochrome in RCSN-3 catecholaminergic cells (8, 21, 24).

10.1021/tx600325u CCC: $37.00 © 2007 American Chemical Society Published on Web 04/11/2007

Inhibition of VMAT-2 and DT-Diaphorase

In the present work, we investigated the role of DT-diaphorase in the neurotoxic action of aminochrome in a cellular model where aminochrome is formed in a more physiological manner in the absence of exogenous oxidizing agents by increasing free cytosolic dopamine when VMAT-2 is inhibited by reserpine in RCSN-3 cells treated with 100 µM dopamine.

Experimental Procedures Chemicals. Dopamine, reserpine, dicoumarol, DME/HAM-F12 nutrient mixture (1:1), and Dulbecco’s phosphate-buffered saline (PBS) were purchased from Sigma Chemical Co. (St Louis, MO). Calcein AM and ethidium homodimer-1 were from Molecular Probes (Eugene, OR). A Thermoscript reverse transcriptasepolymerase chain reaction (RT-PCR) system and Taq DNA polymerase were obtained from Life Technologies (California). Trizol reagent was obtained from Invitrogen (California). The primers were obtained from T-A-G-Copenhagen A/S (Copenhagen, Denmark). Cell Culture. The RCSN-3 catecholaminergic cell line was derived from the substantia nigra of a 4 month old normal Fisher 344 rat. The cloned RCSN-3 cell line showed positive neuronal markers (NSE, synaptophysin, MAP-2, and 200 kDa neurofilament) and completely lacked glial traits (GFAP and S100). All RCSN-3 cells possessed catecholaminergic traits: the presence of TH, melanin, DAT, NET, and strong positive intracellular reaction to paraformaldehyde-glyoxylate (indicating the presence of catecholamine). This cell line also exhibited intracellular Ca2+ increments in response to excitatory neurotransmitter agonists (25). The RCSN-3 cell line grew in monolayers, with a doubling time of 52 h, a plating efficiency of 21%, and a saturation density of 56000 cells/cm2 when kept in normal growth media composed of DME/ HAM-F12 (1:1), 10% bovine serum, 2.5% fetal bovine serum, and 40 mg/L gentamicine sulfate (8, 26). The cultures were kept in an incubator at 37 °C with 100% humidity and an atmosphere of 10% CO2. Transmission Electron Microscopy. After the treatments, RCSN-3 cells were washed three times with PBS, pH 7.4, and fixed in 3% glutaraldehyde for 240 min, washed three times, and postfixed in 2% osmium tetroxide for 60 min at room temperature. The cells were dehydrated in an ascending ethanol battery ranging from 20 to 100%, were later placed in 100% ethanol for 10 min, and were finally embedded in Epon-812 resin. Ultrathin sections were made and impregnated with 4% uranyl acetate and Reynold’s lead citrate. The sections were visualized in a Zeiss EM-900 transmission electron microscope at 50 kV and photographed, the negatives were scanned at 600 ppi × 600 ppi resolution, and the images obtained were analyzed later in a PC-compatible computer using customized software. RT-PCR. The mRNA expression of VMAT was studied in control RCSN-3 cells by using the RT-PCR technique as we described before (8) using the following primers: 5′-ATCCAGACCACCAGACCAGAG-3′and5′-CCCCATCCAAGAGCACCAAGG3′ (27). Cell Death. The cells were incubated with cell culture medium but in the absence of bovine serum and phenol red for 120 min. The concentration used for toxicity experiments was 100 µM dopamine and 0.01, 0.1, 1.0, 10, and 100 µM reserpine in the presence and absence of 100 µM dicoumarol. For control conditions, we used 100 µM dopamine, 1 or 25 µM reserpine, and 100 µM dicoumarol incubated alone. The cells were visualized at 100× magnification in a Nikon Diaphot inverted microscope equipped with phase contrast and fluorescence optics. The toxicity was measured by counting live and dead cells after staining with 2 µM calcein AM and 5 µM ethidium homodimer-1 for 45 min at room temperature. Calcein is a marker for live cells, and ethidium homodimer-1 intercalates in the DNA of dead cells. The cells were counted in a phase contrast microscope equipped with fluorescence, using the following filters: calcein AM, 510-560 nm (excitation), and LP, 590 nm (emission); ethidium homodimer-1, 450-490 nm (excitation) and 515-565 nm (emission).

Chem. Res. Toxicol., Vol. 20, No. 5, 2007 777 Preparation and Inhibition of DT-Diaphorase. DT-diaphorase was prepared by using an azodicoumarol Sepharose-6B affinity column eluted with NADH, resulting in an essentially pure preparation of DT-diaphorase. The concentrated sample was passed through a CM-Sepharose column equilibrated with a solution of 50 mM Tris-HCl, pH 7.0, and 0.25 M sucrose in order to remove minor contamination by two proteins with high isoelectric points as described by Segura-Aguilar et al. (28). The DT-diaphorase activity of pure enzyme was assayed in 50 mM Tris-HCl (pH 7.5), 0.08% Triton X100, 500 µM NADH as electron donors, 10 µM menadione as acceptor, and 77 µM cytochrome c by following the absorbance change of cytochrome c as a secondary acceptor at 550 nm (28). The inhibition of DT-diaphorase was measured by using a constant concentration of VMAT inhibitor reserpine (60 µM) and increasing the concentration of NADH up to 500 µM. The inhibitorbinding constant (Ki) of reserpine on DT-diaphorase activity was calculated by a Lineweaver-Burk plot. Immunocytochemistry. The coverslips containing control RCSN-3 cells at 50% confluence were washed twice with PBS, pH 7.4. They were then fixed for 30 min with methanol at -20 °C. The cells were rinsed twice with PBS and blocked with 1.5% bovine albumin serum diluted in PBS for 40 min. The coverslips were incubated with the primary antibody (rabbit anti-VMAT-2, Novocastra, New Castle Upon Tyne, United Kingdom) at a dilution of 1:40 in PBS overnight. After washing, the cells were incubated with a secondary antibody [biotinylated anti-mouse IgG (H + L), Vector Laboratories] diluted 1:250 in PBS for 1 h. The cells were later incubated with Cy-3-conjugated streptavidin (3 µg/mL) (Jackson Inmunoresearch Laboratories) for 1 h. The streptavidin solution was then removed, and the cells were washed three times with PBS. Coverslips were mounted onto slides with DAKO fluorescent mounting medium and kept in the dark at 4 °C. Data Analysis. All data were expressed as means ( SD values. The statistical significance was assessed using analysis of variance (ANOVA) for multiple comparisons and Student’s t test.

Results We have used a catecholaminergic cell line (RCSN-3) derived from adult rat substantia nigra to study the role of DT-diaphorase in aminochrome-induced cell death. For this proposition, we have inhibited the VMAT-2 with reserpine in order to induce intracellular dopamine oxidation to aminochrome by increasing the concentration of free cytosolic dopamine. To decide whether RCSN-3 cells were a suitable model cell line for this study, we evaluated whether these cells formed melanin since aminochrome resulting from dopamine autoxidation is one of the precursors of neuromelanin. In the cytoplasm of RCSN-3 cells treated with 100 µM dopamine, we observed a double membrane vacuole containing a dark pigment (Figure 1A,B) that is similar to melanin-containing bodies described before (16). The formation of melanin bodies was followed by endocytosis into phagocytic vacuoles (Figure 1C). The expression of VMAT-2 mRNA in RCSN-3 catecholaminergic cells was demonstrated by using RT-PCR (Figure 2A), and the expression of VMAT-2 protein was determined by using immunocytochemistry (Figure 2B). We have incubated RCSN-3 cells with reserpine between 25 and 100 µM for 2 h to study the possible neurotoxic effects of reserpine alone. At 25 µM reserpine, a moderate cell death was observed (22 ( 4% cell death; Figure 3A); therefore, we have used this concentration to study one-electron reduction of aminochrome in RCSN-3 cells by incubating the cells in the presence of dicoumarol, an inhibitor of DT-diaphorase. We incubated the cells with 100 µM dopamine and 25 µM reserpine, resulting in a 53 ( 4% cell death. Because DT-diaphorase prevents one-electron reduction of aminochrome to leukoaminochrome o-semiquinone radical, we expected that incubation

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Figure 1. Transmission electron microscopy on RCSN-3 cells. Transmission electron microscopy of RCSN-3 cells incubated for 6 h with 100 µM dopamine reveals the presence of a body containing neuromelanin (A), and we observe the double membrane of the melanin body (B; white arrow). A phagocytic vacuole under endocytosis of a melanin body is shown with the withe arrow (magnification of 30000×; C). The magnifications of A and C were 30000× and 50000× in B.

of RCSN-3 cells with 100 µM dopamine, 25 µM reserpine, and 100 µM dicoumarol would dramatically increase the cell death. However, under these conditions, the cell death showed only a moderate, and no significant, increase (67 ( 9%). The cell deaths under incubation with cell culture media, 100 µM dopamine, 100 µM dicoumarol, or 100 µM dopamine with 100 µM dicoumarol were 3 ( 3, 6 ( 2, 3 ( 2, and 12 ( 5%, respectively (Figure 3B). The lack of effect of inhibition of DT-diaphorase by dicoumarol, when the cells were incubated with dopamine and reserpine, opens the question about a possible unknown action of reserpine as an inhibitor of DT-diaphorase. To determine whether reserpine acts as an inhibitor of DT-diaphorase, we measured its enzyme activity in vitro with pure enzyme using menadione-cytochrome c assay in the presence of a constant concentration of the enzyme, reserpine (60 µM), and an increasing concentration of NADH. The results showed that reserpine is a competitive inhibitor of DT-diaphorase with an inhibitor-binding constant (Ki) of 24 µM (Figure 4). We tested the effect of reserpine on cell death with lower concentrations than 24 µM (Ki for DT-diaphorase) during 2 h of incubation in order to determine a reserpine concentration to study the role of DT-diaphorase. A 10 µM concentration of reserpine induced 11 ( 6% cell death, but 1 µM reserpine induced only 3 ( 1% cell death (Figure 5A). We used the

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Figure 2. VMAT-2 expression in RCSN-3 cells. (A) VMAT-2 mRNA expression was determined by RT-PCR. (B) VMAT-2 protein expression was determined by immunohistochemistry.

concentration of 1 µM reserpine to measure the cell death in the presence of dicoumarol at 2, 6, and 12 h since this concentration does not inhibit DT-diaphorase but is severalfold higher than the IC50 required to inhibit 50% VMAT-2 transport activity. When the cells were incubated with 1 µM reserpine and 100 µM dopamine or 1 µM reserpine, 100 µM dopamine, and 100 µM dicoumarol, no significant cell death was observed at 2 and 6 h. However, a significant cell death was observed at 12 h when the cells were incubated with 1 µM reserpine, 100 µM dopamine, and 100 µM dicoumarol (Figure 5). We used transmission electron microscopy to determine morphological changes and to define cell death characteristics in RCSN-3 cells incubated with 1 µM reserpine, 100 µM dopamine, and 100 µM dicoumarol for 6 h. Under these conditions, we observed (i) the formation of blebbing; (ii) chromatin condensation accompanied with the formation of massive patches in contact with the nuclear membrane; and (iii) the smoothness of the cell’s surface, that is, the lack of surface microprojections (Figure 6F,G). However, in RCSN-3 cells incubated with serum and phenol red-free essential culture medium in the absence or presence of 1 µM reserpine, 100 µM dopamine, 100 µM dicoumarol, 1 µM reserpine, or 1 µM reserpine with 100 µM dopamine for 6 h, we did not observe structural changes such as blebbing formation, chromatin condensation, and formation of massive patches in the perinuclear area. In addition, we observed the existence of surface microprojections and microvilli (Figure 6A-E, respectively).

Inhibition of VMAT-2 and DT-Diaphorase

Figure 3. Effect of reserpine and dicoumarol on RCSN-3 cells. The effect of reserpine between 0 and 100 µM on RCSN-3 cells incubated for 2 h (A). (B) To study the effect of DT-diaphorase inhibition with dicoumarol in RCSN-3 cells treated with reserpine, the cells were incubated for 2 h with 100 µm dopamine (DA); 25 µM reserpine (R); 100 µM dicoumarol (D); 100 µM dopamine and 25 µM reserpine (DAR); 100 µM dopamine and 100 µM dicoumarol (DAD); and 25 µM reserpine and 100 µM dicoumarol (DARD). The values are the means ( SD (n ) 3 and the statistical significance was assessed using ANOVA for multiple comparisons and Student’s t test; ***p < 0.001 and n ) 3).

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Figure 5. Cell death induced by reserpine and dicoumarol in RCSN-3 cells. (A) The effect of increasing concentrations of reserpine (0-100 µM) was studied on RCSN-3 cells. (B) The cells were incubated cell culture medium (C) in the presence of 100 µM dopamine (DA); 1 µM reserpine (R); 100 µM dicoumarol (D); 100 µM dopamine and 1 µM reserpine (DAR); 100 µM dopamine and 100 µM dicoumarol (DAD); 100 µM dopamine, 1 µM reserpine, and 100 µM dicoumarol (DARD); 100 µM dicoumarol and 1 µM reserpine (DR); and 100 µM dopamine, 1 µM reserpine, and 100 µM dicoumarol (DARD) for 2, 6, and 12 h. The values are the means ( SD (n ) 3 and the statistical significance was assessed using ANOVA for multiple comparisons and Student’s t test; **p < 0.01 and n ) 3).

disruption of the cristae architecture, which remains closely packed when the cells were treated with 1 µM reserpine, 100 µM dopamine, and 100 µM dicoumarol for 6 h (Figure 7F, inset). These observations contrast with the normal cristae architecture and membrane integrity in RCSN-3 cells incubated with serum and phenol red-free medium in the absence or presence of 1 µM reserpine with essential minimal cell culture medium, 100 µM dopamine, 100 µM dicoumarol, 1 µM reserpine, or 1 µM reserpine with 100 µM dopamine for 6 h (Figure 7A-E, respectively).

Discussion Figure 4. Determination of reserpine inhibitior-binding constant on DT-diaphorase. The reciprocal of the initial rate of DT-diaphorase activity vs the reciprocal of the substrate concentration (NADH) was plotted in the absence and presence of 60 µM reserpine. The incubation mixture contained 5 µM menadione, 77 µM cytochrome, 30 ng of DTdiaphorase, and increasing concentrations of NADH between 0 and 500 µM.

We observed the alteration of mitochondria ultrastructure such as (i) disorganization of the mitochondrial matrix due to separation of the outer membrane from the internal membrane and considerable enlargement of the intermembrane space; (ii) disruption of the external mitochondrial membrane; and (iii)

The molecular structure of dopamine allows a double role in the nervous system: to be an essential neurotransmitter and a toxic substance that determines apoptosis in different cell lines (29-38). One possible explanation for the ability of dopamine to act as a neurotoxin is its ability to oxidize to aminochrome and induce neurotoxicity by (i) generation of a potent neurotoxin (leukoaminochrome o-semiquinone radical) during one-electron reductive metabolism (8, 21, 22, 24) and/or (ii) adduct formation with R-synuclein (17) generating neurotoxic protofibrils. The uptake of dopamine into monoaminergic vesicles seems to be a very important mechanism to prevent dopamine oxidation (16). The low pH inside vesicles prevents dopamine oxidation to

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Figure 6. Transmission electron microscopy of RCSN-3 cells treated for 6 h. We can observe the cell’s surface with microprojections and microvilli (M with black arrows) not only in control cells incubated with serum and phenol red-free culture medium alone (A) but also in cells treated with 100 µM dopamine (B), 1 µM reserpine (C), 100 µM dicoumarol (D), and 100 µM dopamine and 1 µM reserpine (E). The black arrowhead shows the smoothness of the cell’s surface, that is, the lack of surface microprojection and microvilli, and the black arrow shows the membrane blebbing in cells treated with 100 µM dopamine, 1 µM reserpine, and 100 µM dicoumarol (F and its insert). The white arrow shows the formation of massive patches of chromatin in contact with the nuclear membrane when the cells were treated with 100 µM dopamine, 1 µM reserpine, and 100 µM dicoumarol (F). The inset of F shows a cell with several blebbing. The magnification was 3000× in all of the pictures. The control was incubated with cell culture medium alone (A).

aminochrome, as the hydroxyl groups in the molecule are protonated, yielding oxidation or polymerization very unlikely. It has been reported that neuromelanin synthesis is abolished by adenoviral-mediated overexpression of the synaptic VMAT2, which would decrease cytosolic dopamine by increasing vesicular accumulation of the neurotransmitter (16). Another mechanism that prevents dopamine autoxidation to aminochrome is dopamine catabolism by MAO. In this regard, overexpression of MAO A induces protection against intracellular L-DOPA toxicity (39). However, despite the existence of these two mechanisms to prevent dopamine oxidation to aminochrome, we can observe the existence of neuromelanin in the substantia nigra, which accumulates as a function of age in human substantia nigra (10). To study the mechanism of aminochrome toxicity and the neuroprotective role of DT-diaphorase, we incubated RCSN-3 cells with dopamine and reserpine, a VMAT-2 inhibitor, to increase free cytosolic dopamine and thereby increase dopamine oxidation to aminochrome. We used RCSN-3 cells as a model cell line since this is a catecholaminergic cell line with a dopamine transporter, tyrosine hydroxylase expression, and melanin formation and these cell cultures have been used before

to study aminochrome toxicity (8, 21, 22, 24, 40). The presence of double membrane melanin bodies, when the RCSN-3 cells were incubated with 100 µM dopamine, supports the idea that dopamine oxidation to aminochrome occurs in RCSN-3 cells under the conditions used during our experiments (Figure 1). Bush et al. (12) have shown by employing sophisticated physical methods that melanin granule is formed by a pheomelanin at the core and eumelanin at the surface. We have not determined the composition of the observed pigmented aggregates in RCSN-3 cells; therefore, we do not consider these aggregates as neuromelanin. The increased oxidative stress with concomitant formation of H2O2 produced by one-electron reduction of aminochrome to leukoaminochrome o-semiquinone radical, when DT-diaphorase is inhibited, may result in the disruption of double membrane granules, resulting in degradation of the eumelanic surface of neuromelanin, thus exposing the pheomelanic core that behaves as a prooxidant itself (41). The oxidation potential of neuromelanin is not able to reduce oxygen, but the degradation of the eumelanin surface in the neuromelanin exposes pheomelanin to the surface, which has an oxidation potential that it is able to reduce oxygen (12). Neuromelanin has been reported to inhibit 26S proteasome activity (42, 43).

Inhibition of VMAT-2 and DT-Diaphorase

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Figure 7. Effect of reserpine and dicoumarol on RCSN-3 cell’s mitochondria. RCSN-3 cell line treated with 100 µM dopamine (B), 1 µM reserpine (C), 100 µM dicoumarol (D), 100 µM dopamine and 1 µM reserpine (E), and 100 µM dopamine, 1 µM reserpine, and 100 µM dicoumarol (F) incubated for 6 h in the absence of bovine serum and phenol red. The control cells incubated with serum and phenol red-free culture medium alone (A). The magnification was 30000× in all of the pictures. The black arrows show damaged mitochondria.

It is reasonable to think that the dopamine oxidation to aminochrome and the subsequent polymerization to melaninic compound are normal processes due to the presence of DTdiaphorase activity, which prevents the aminochrome conversion to neurotoxic species generated by one-electron reduction. An interesting and new finding is that the inhibitor of VMAT2, reserpine, is also an inhibitor of DT-diaphorase, explaining the lack of effect of dicoumarol when the cells are incubated with 25 µM reserpine since the inhibitior-binding constant for DT-diaphorase is 24 µM. Therefore, to study the role of DTdiaphorase inhibited by dicoumarol, we used a concentration of 1 µM reserpine, a concentration several-fold over the reserpine IC50 (4-25 nM), which is required to inhibit 50% of VMAT-2 transport activity (44, 45). Interestingly, no significant cell death was observed when the cells were incubated with 1 µM reserpine, 100 µM dopamine, and 100 µM dicoumarol for 2 and 6 h, in contrast with results obtained in the same cell line but using metals such as Mn3+, Cu2+, and Fe3+ as oxidizing agents of dopamine (8, 21, 22, 24). Our result suggests that metals (Mn3+, Cu2+, and Fe3+) are responsible for aminochrome acute toxicity observed in RCSN-3 cells incubated for 2 h. The experimental model presented in this work could be more physiological than the use of metals to induce dopamine autoxidation since it seems to be plausible that dopamine

oxidation to aminochrome and its polymerization to neuromelanin in vivo is driven by the existence of free dopamine in dopaminergic neurons. In the present model experiment, we observed significant cell death only at 12 h, suggesting that this cell death was probably apoptotic. Electron transmission microscopy results support this idea since we observed several signals of apoptosis such as formation of blebbing, chromatin condensation accompanied by the formation of massive patches in contact with the nuclear membrane, and the smoothness of the cell’s surface, that is, the lack of surface microprojections. The cells start to show protrusions of the plasma membrane, commonly referred to as blebs or membrane blebbing, when microvilli and microprojections are lost. The cells shrink, and finally, the blebs separate (Figure 6F, inset), forming apoptotic bodies densely packed with cellular organelles and nuclear fragments that are engulfed by phagocytosis of surrounding cells. Blebbing of the plasmatic membrane is considered a morphological characteristic of cell death by apoptosis. Dopamine-induced cell death has been reported to be a apoptotic process, which is in agreement with our results, due to the fact that it seems plausible that dopamineinduced apoptotic cell death is mediated by dopamine oxidation to aminochrome (38, 46, 47).

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catalyzing one-electron reduction of quinones to semiquinones radicals. However, only DT-diaphorase catalyzes two-electron reduction of quinones to hydroquinones (49) and constitutes the 97% total quinone reductase activity in rat substantia nigra (23). Inhibition of DT-diaphorase by dicoumarol allows one-electron reduction of aminochrome, with the formation of leukoaminochrome o-semiquinone radical, which is a very reactive species that autoxidizes by reducing oxygen to superoxide radicals, thus initiating a redox cycling process (18, 19). In this process, minimal aminochrome amounts can produce increased amounts of free radicals even though all oxygen has been reduced or all NAD(P)H has been oxidized (18). When DTdiaphorase is inhibited by dicoumarol, aminochrome cannot form melanin due to the fact that aminochrome is reduced via one-electron by flavoenzimes, which induce cell death instead of melanin formation, since we did not observe an increase of melanin formation under these conditions. The results presented in this study strongly support the neuroprotective role of DT-diaphorase against the neurotoxic effects of one-electron reduction of aminochrome (Figure 8) and suggest that RCSN-3 cells incubated with 1 µM reserpine and 100 µM dopamine seem to be an excellent experimental model cell line to study the mechanism involved in dopamine oxidation and toxicity. These results support the idea that melanin itself does not induce neurotoxicity. Another interesting finding is that reserpine is not only an inhibitor of VMAT-2 but also an inhibitor of DT-diaphorase at higher concentrations. Figure 8. Possible mechanism for reserpine- and dicoumarol-induced cell death in RCSN-3 cells. Reserpine inhibits dopamine transport into vesicles driven by VMAT-2 resulting in an increase of free cytosolic dopamine, which autoxidizes to aminochrome the precursor of neuromelanin. Aminochrome into the cell has four alternatives to react: (i) aminochrome polymerization to neuromelanin, (ii) to form adducts with R-synuclein stabilizing the neurotoxic protofibrils, (iii) one-electron reduction of aminochrome to leukoaminochrome o-semiquinone radical catalyzed by flavoenzymes with the unique exception of DT-diaphorase, and (iv) two-electron reduction of aminochrome, which prevents neurotoxic pathways, one-electron reduction of aminochrome, and formation of R-synuclein-aminochrome adducts. One-electron reduction of aminochrome and/or formation of R-synuclein-aminochrome adducts, which stabilizes neurotoxic protofibrils, induces mitochondrial damage followed by a apoptotic process resulting in cell death.

Another feature of aminochrome-induced cell death is mitochondrial damage since the mitochondria cristae architecture and membrane are disrupted after RCSN-3 cells are incubated with 1 µM reserpine, 100 µM dopamine, and 100 µM dicoumarol for 6 h. The main finding is the focal rupture of the external mitochondrial membrane and the protrusion of the internal membrane through an opening that was produced in the external membrane to produce a hernia. In the region of the mitochondria where the external membrane is still intact, the cristae remained close packed, and the hernia can be considered as a vesicle with very few or with no cristae (Figure 7F, inset). We suggest that these mitochondrial alterations could represent preapoptotic lesions that are manifested prior to this process of cell death, in agreement with the results reported by CarranzaRosales et al. (48). Our results support the proposed hypothesis that DTdiaphorase plays a neuroprotective role in dopaminergic neurons by preventing one-electron reduction of aminochrome to leukoaminochrome o-semiquinone radical (8, 18, 21). A significant cell death was observed only when RCSN-3 cells were incubated with reserpine and dopamine together with the DT-diaphorase inhibitor dicoumarol. All flavoenzimes found in the cytosol in the glycolysis or in the mitochondria (ubiquinone reductase, NADH cytochrome c reductase) may act as quinone reductase,

Acknowledgment. This work was supported by FONDECYT Grant 1061083 (Chile). We thank Nancy Olea and collaborators from the Electronic Microscopy Unit from the Faculty of Medicine, University of Chile.

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