Introduction

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Methylcyclopentadienyl manganese tricarbonyl (MMT), an organic manganese-containing compound, has been recently legalized for use as a lead replacement in fuels in the United States and is marketed as HITEC 3000 or AK-33X and contains around 25% manganese (Zayed et al., 1994; Frumkin and Solomon, 1997). In the past, the major health effects of MMT have centered on possible exhaust products and ambient particulates caused by MMT combustion. Excessive manganese exposure has been reported to cause Parkinsonian-like symptoms, known as Manganism (Calne et al., 1994). Although Manganism differs from Parkinson's disease (PD) in neuropathology, clinical presentations of each disease are similar (Aschner, 2000). Furthermore, numerous epidemiological studies have demonstrated a positive association between environmental risk factors and increased incidence of idiopathic, geriatric onset PD (Veldman et al., 1998; Tanner et al., 1999). The results of these studies demonstrated that no significant genetic correlate exists in geriatric onset PD, which by implication suggests an environmental factor, and may contribute to the promotion of the disease. Considering the epidemiological and toxicological evidence collectively, MMT and its manganese combustion products may be considered as potential environmental risk factors for PD and its related disorders.

The manganese atom of MMT is linked to a methylcyclopentadiene ring and three carbonyl groups, and these organic substitutions make MMT highly lipophilic, which might increase the bioaccumulation of this organometallic compound. Recently, a comparative toxicokinetic study in rats has demonstrated that the MMT-derived manganese accumulates in plasma 37 times more than inorganic manganese along with a slower clearance rate (Zheng et al., 2000). Gianutsos and Murray (1982) have also demonstrated MMT-induced dopamine depletion in rat brain, which is suggestive of deleterious cytotoxic effects. Concern has recently been expressed over toxicity of MMT itself due to the possibility of exposure through dermal absorption from accidental spills, deliberate use of gasoline as a solvent cleaner, and solvent abuse such as intentional gasoline fume inhalation (Zayed et al., 1994). Furthermore, Garrison et al. (1995) argue that critical exposure sources for MMT do not include engine exhausts, but instead accidental releases during manufacture, handling, transportation, and storage as most likely sources for environmental and human exposure.

Apoptosis, the presumed mechanism of nigrostriatal cell death in PD (Hirsch et al., 1999; Offen et al., 2000), can be initiated by either receptor-stimulated (e.g., Fas-ligand-mediated) or toxicant-induced pathways. Both signal pathways share a mitochondrial link to downstream apoptotic events, the specifics of which can vary by initiator stimulus and cell type. A common link between varying detail of apoptotic pathways is the role of Bcl-2 as an inhibitory lock on apoptosis (Voehringer and Meyn, 2000). Overexpression of Bcl-2 in in vitro cell line studies has been shown to inhibit cell death and reduce reactive oxygen species (ROS) generation, which directly implicates mitochondria as prime targets in apoptotic cell death.

The present study entails a detailed assessment of MMT-mediated toxic effects in dopamine-producing PC-12 cells and extends work reported by us previously (Anantharam et al., 2002). PC-12 cells have proven to be an in vitro experimental model of choice to study effects of various neurotoxic agents, including 6-hydroxydopamine, MPP⁺, paraquat, and manganese on dopaminergic cells (Shafer and Atchison, 1991; Desole et al., 1997b; Li and Sun, 1999; Viswanath et al., 2001; Anantharam et al., 2002; Park et al., 2002). PC-12 cells are electrically excitable and neurosecretory (dopamine, norepinephrine, and/or acetylcholine), and contain many membrane-bound and cytosolic macromolecules associated with neurons (Shafer and Atchison, 1991). In the present study, we have examined cytotoxicity, neurotransmitter depletion, ROS generation, caspase-3 activation, depolarization of mitochondrial membrane potential, and DNA fragmentation as toxicological endpoints during acute exposure of MMT to delineate the early cellular events that might contribute to the degenerative process in dopamine-producing cells. Furthermore, we demonstrated protective effects of Bcl-2 protein overexpression, antioxidants, and dopamine synthesis and catabolism inhibitors toward reduction of MMT-induced ROS generation, caspase-3 activation, and DNA fragmentation, suggesting causal roles of mitochondrial dysfunction, oxidative stress, and dopamine catabolism in MMT-induced cytotoxicity.

	Materials and Methods

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Chemicals. MMT (Aldrich Chemical Co., Milwaukee, WI) is a viscous yellow liquid with an herbaceous odor. MMT was prepared fresh and dissolved in dimethyl sulfoxide. Trolox was purchased from Aldrich Chemical Co. Manganese(II) chloride, deprenyl, -methyl-L-p-tyrosine (-MPT), cytosine arabinoside, and N-acetyl-L-cysteine (NAC) were obtained from Sigma-Aldrich (St. Louis, MO). Caspase-3 substrate, Ac-DEVD-AMC, was purchased from Bachem Biosciences (King of Prussia, PA). Cell culture media and reagents were purchased from Invitrogen (Carlsbad, CA). Dihydroethidine, dichlorofluorescein-diacetate, acridine orange, and DiOC₆ were purchased from Molecular Probes (Eugene, OR). Other routine laboratory reagents were obtained from Fisher Scientific (Pittsburgh, PA).

Cell Culture. Dopamine-producing PC-12 cells were obtained from the American Type Culture Collection (Rockville, MD). Nondopaminergic cells were used in the cytotoxicity experiments to determine the differential toxic response of MMT in dopamine-producing cells versus nondopaminergic cells. Striatal -aminobutyric acidergic cells (M213-20 cells) were a generous gift from Dr. William J. Freed (National Institute on Drug Abuse, Cellular Neurobiology Branch, Baltimore, MD). PC-12 cells were grown in RPMI 1640 medium containing 10% heat-inactivated horse serum, 5% fetal bovine serum, 2 mM L-glutamine, 50 units penicillin, and 50 µg/ml streptomycin. M213-20 cells were grown in DMEM containing 10% fetal bovine serum, 2 mM L-glutamine, 50 units penicillin, and 50 µg/ml streptomycin. Human Bcl-2-transfected PC-12 (PC-12HB2-3) and vector-transfected (PC-12V4) cells were generous gifts from Drs. Yutaka Eguchi and Yoshihide Tsujimoto (Osaka University, Osaka, Japan). PC-12V4 and PC-12HB2-3 cells were grown in DMEM with 7% horse serum and 4% fetal bovine serum. PC-12 cells were placed in 75-cm² cell culture flasks at 37°C under a humidified atmospheric condition of 5% CO₂ and 95% air, and 3- to 6-day-old cells were used for the experiments. M213-20 cells were grown at 33°C in 5% CO₂ incubator. Cells were suspended in either Krebs-Ringer solution (125 mM NaCl, 5 mM KCl, 25 mM HEPES, 6 mM glucose, 5 mM NaHCO₃, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, and 2.4 mM CaCl₂, pH 7.4), serum-free RPMI 1640 medium, or DMEM at a concentration of 2 × 10⁶ cells/ml, depending upon the biochemical assay method.

Treatment Paradigm. PC-12 and M213-20 cells were treated with MMT (0-1000 µM) dissolved in dimethyl sulfoxide (final concentration in incubates <0.5%). After 1-h incubation with MMT at 37°C, dead and live cells were determined by trypan blue exclusion method using an improved Neubauer hemocytometer. Pretreatment with -MPT was performed 24 h before MMT treatment, whereas pretreatments with NAC, deprenyl, or Trolox were performed 30 min before MMT exposure. The cell viability was normalized as percentage of vehicle control.

Lactate Dehydrogenase Assay. LDH activity in the cell-free extracellular supernatant was quantified as an index of acute cell death in a 96-well format (Kitazawa et al., 2001). Extracellular supernatant (10 µl) was added to 200 µl of 0.08 M Tris buffer, pH 7.2, containing 0.2 M NaCl, 0.2 mM NADH, and 1.6 mM sodium pyruvate. LDH activity was measured continuously by monitoring the decrease in the rate of absorbance at 339 nm using a microplate reader (Molecular Devices, Sunnyvale, CA), and the temperature was maintained at 37°C during reading. Changes in absorbance per minute (A/T) were used to calculate LDH activity (U/I), using the following equation: U/I = (A/T) × 9682 × 0.66, where 9682 is a coefficient factor, and 0.66 is a correction factor at 37°C. Activity was corrected per milligram of protein and expressed as percentage of total LDH signal.

Neurotransmitter Determinations. Extracellular and intracellular dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) were measured by high-performance liquid chromatography with electrochemical detection (HPLC-EC) as described previously, with slight modification (Kanthasamy et al., 1991). Briefly, PC-12 cells were resuspended in Krebs-Ringer solution at a density of 3 to 7 × 10⁶ cells/ml. MMT was added, and cells were incubated in a shaking water bath for 2 h at 37°C. Cells were then centrifuged at 1500g for 15 min, and supernatants were collected and stored with antioxidant solution (15 mM Na₂EDTA, 50 mM Na₂S₂O₅, and 4 mM HClO₄). Cell pellets were resuspended in antioxidant solution and allowed to lyse for 15 min on ice. Samples were stored at 80°C until further analysis. Before analysis by HPLC-EC, samples were centrifuged at 10,000g for 15 min at 4°C to pellet cellular debris.

ROS Assay. We used dichlorofluorescein-diacetate (DCF-DA) to measure ROS by fluorometric assay. Briefly, the cells were harvested and washed in Krebs-Ringer solution, resuspended at 1 to 3 × 10⁶ cells/ml, and loaded with DCF-DA (15 min, 37°C). Cells were pelleted, the supernatant containing the excess fluor was removed, and the pellet was then resuspended in 2 ml of Tris buffer, pH 7.4. Production of 2,7-dichlorofluorescein (DCF-H), a fluorescent product of hydrolyzed DCF-DA, was monitored over 1 h by spectrofluorometer (488/525 nm). MMT was added to the tubes, vortexed, and transferred to cuvettes for fluorescent readings [F(0)]. After the readings were obtained, the samples were transferred back to the tube and placed in a water bath for 60-min incubation. After 60 min, the tubes were removed and the fluorescence was read again as the F(60) endpoint measurement. Data were expressed as percentage of vehicle control.

Detection of Mitochondrial Membrane Potential. Depolarization of mitochondrial membrane potential (m) was assessed by flow cytometric analysis using DiOC₆ (Kitazawa et al., 2001). DiOC₆ (40 nM) was added to incubation medium 15 min before the end of treatment period, and the incubation continued at 37°C. Then the cells were washed once, resuspended in phosphate-buffered saline, pH 7.4, and analyzed by flow cytometry with excitation at 484 nm and emission at 501 nm using a flow cytometer. Data were analyzed by CellQuest software (BD Biosciences).

Caspase-3-Like Activity Assay. Caspase-3 activity was determined by following procedure described previously, with slight modification (Yoshimura et al., 1998). Briefly, after the exposure to MMT, cells were washed once with phosphate-buffered saline, pH 7.4, and resuspended in lysis buffer containing 50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 10 mM EGTA, and 10 µM digitonin. Cells were then incubated at 37°C for 20 min to allow complete lysis. Lysates were quickly centrifuged, and cell-free supernatants were incubated with 50 µM Ac-DEVD-AMC (caspase-3 substrate) at 37°C for 1 h. Formation of 7-amino-4-methylcoumarin (AMC), as a result of cleavage of substrates by caspase-3, was measured by spectrofluorometer (Molecular Devices) with excitation at 380 nm (slit width 10 nm) and emission at 460 nm (slit width 20 nm). Caspase activity was expressed as fluorescence units per milligram of protein per hour of incubation. Protein content of samples was measured using the Bradford protein assay reagent (Bio-Rad, Hercules, CA).

DNA Fragmentation Assay. DNA fragmentation assay was performed using the Cell Death Detection ELISA Plus Assay kit (Roche Applied Science, Indianapolis, IN). This kit measures amount of histone-associated low molecular weight DNA in the cytoplasm of cells and has recently been used in quantitation of apoptosis because of its reliability and high sensitivity (Anantharam et al., 2002). PC-12 cells in antioxidant studies were pretreated with antioxidant for 30 min. Cells were then exposed to 200 µM MMT for 1 h. After MMT treatment, cells were pelleted at 200g for 5 min and washed once with phosphate-buffered saline, pH 7.4. Cells were then incubated with a lysis buffer (supplied with the kit) at room temperature. After 30 min, samples were centrifuged and 20-µl aliquots of the supernatant were then dispensed into streptavidin-coated 96-well microtiter plates followed by addition of 80 µl of antibody cocktail and incubated for 2 h at room temperature with mild shaking. The antibody cocktail consisted of a mixture of anti-histone biotin and anti-DNA-HRP directed against various histones and antibodies to both single-stranded DNA and double-stranded DNA, which are major constituents of the nucleosomes. After incubation, unbound components were removed by washing with the incubation buffer supplied with the kit. Quantitative determination of the amount of nucleosomes retained by anti-DNA-HRP in the immunocomplex was determined spectrophotometrically with 2,2'-azino-di[3-ethoxybenzyl thiazoline sulfonate] as an HRP substrate (supplied with the kit). Measurements were made at 405 nm against a 2,2'-azino-di[3-ethoxybenzyl thiazoline sulfonate] solution as a blank (reference wavelength 490 nm) using a Spectramax microplate reader (Molecular Devices).

Western Blot Analysis of Bcl-2 Expression. PC-12 cells were centrifuged at 200g for 5 min. Cell pellets were then washed once with ice-cold Ca²⁺-free phosphate-buffered saline and resuspended in 2 ml of homogenization buffer (20 mM Tris-HCl, pH 8.0, 10 mM EGTA, 2 mM EDTA, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 25 µg/ml aprotinin, and 10 µg/ml leupeptin). Suspensions were sonicated for 10 s and centrifuged at 100,000g for 1 h at 4°C. Supernatants were discarded and pellets were gently resuspended in ice-cold Ca²⁺-free phosphate-buffered saline. Protein concentration of each sample was determined using the Bradford protein assay reagent.

In Situ Assessment of Apoptosis. Changes in nuclear morphology and DNA conformation of MMT-treated cells were assessed qualitatively with fluorescent DNA-binding dyes acridine orange. Acridine orange exhibits metachromatic fluorescence that is sensitive to DNA conformation. Apoptotic cells stained with acridine orange show reduced green and enhanced red fluorescence in comparison with normal cells (Kitazawa et al., 2001). PC-12 cells were grown on laminin (5 µg/ml)-coated slides for 2 to 3 days in a 37°C, 5% CO₂ incubator. Cells were washed twice with phosphate-buffered saline, pH 7.4, and treated for 1 h with MMT (200 µM). Cells were incubated with 10 µM acridine orange for 15 min at room temperature in the dark. Cells were again washed with phosphate-buffered saline, mounted with coverslips, and observed under a DiaPhot microscope (Nikon, Tokyo, Japan) with attached SPOT digital camera (Diagnostic Instruments, Sterling Heights, MI).

Data Analysis and Statistics. Data are expressed as mean ± S.E.M., and statistical significance was determined by analysis of variance with either Dunnett's test in the case of multiple comparisons with control or Tukey-Kramer means separation test for multiple comparisons between treatment groups. Single comparisons were performed by Student's t test or Welch-corrected unpaired t test where appropriate. Differences were accepted as significant at p < 0.05 or less.

	Results

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MMT Decreases Cell Viability. PC-12 cells were exposed to 0 to 1000 µM MMT for 1 h and cell viability was measured by trypan blue dye exclusion. Figure 1A shows the relationship between MMT concentration (log µM) and percentage of cell survival relative to the control. Exposure of PC-12 cells to various concentrations of MMT resulted in a concentration-dependent decrease in cell viability, and the EC₅₀ of MMT was calculated to be approximately 206 µM by three-parameter nonlinear regression. Based upon these results, subsequent measurement of various biochemical indices in key mechanistic studies were performed in PC-12 cells treated with 200 µM MMT. Treatment with as high as 1 mM inorganic Mn²⁺ did not show any significant alteration in cell viability in PC-12 cells for 1-h exposure, and more than 24 h was required to induce cytotoxicity in PC-12 cells (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Effect of MMT on cell viability in dopamine-producing and nondopaminergic cells. A, MMT-induced loss of cell viability after 1-h treatment in dopamine-producing PC-12 cells () and M213-20 () cells was determined by trypan blue exclusion method and was analyzed by three-parameter logistic regression (EC50 and 95% confidence interval of EC50: PC-12 cells, 205.8 µM, 99.9-424.2; and M213-20 cells, 590.5 µM, 453.4-769.0). EC50 values of PC-12 and M213-20 cells were significantly different (p < 0.02, Welch-corrected unpaired t test). B, cytotoxicity of MMT in cerebellar granule cells was determined by measurement of extracellular LDH activity after 1 h of treatment. "U" denotes untreated cerebellar granule cells. Data represent results of at least three separate experiments in triplicate and are expressed as percentage of vehicle-treated group (mean ± S.E.M.). , p < 0.05 compared with vehicle-treated group.

MMT Causes Dopamine Depletion. Treatment of PC-12 cells with MMT (0-1000 µM) for 1 h resulted in a significant concentration-dependent depletion of intracellular dopamine (Fig. 2A; p < 0.0001). Depletion of dopamine by MMT in PC-12 cells seemed to be biphasic, with an initial depletion of ca. 44% at concentrations of 30 to 100 µM MMT, depleting dopamine further with increasing MMT concentration to >95% depletion at concentrations of MMT above 100 µM (Welch-corrected unpaired t test, p < 0.02; 300 versus 100 µM MMT). Extracellular dopamine concentrations did not increase across the concentration range of MMT used herein, suggesting that MMT does not promote dopamine secretion. Extracellular DOPAC increased 2.3-fold and seemed to increase inversely to cellular dopamine depletion, whereas intracellular DOPAC did not change significantly across the concentration range of MMT used herein (Fig. 2B). It is presently unclear whether tyrosine hydroxylase inhibition contributes to observed dopamine depletion because of the short time (1-h) exposure of MMT. Further studies are needed to determine the exact neurochemical mechanisms underlying MMT-induced dopamine depletion and DOPAC formation.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Dopamine depletion in MMT-treated PC-12 cells. Intracellular and extracellular dopamine (A) and DOPAC (B) levels were determined by HPLC-EC after 1-h treatment of PC-12 cells with MMT. Significant increases in extracellular DOPAC and decreases in intracellular dopamine were analyzed by analysis of variance followed by a Dunnett's test (dopamine, p < 0.0001; and DOPAC, p < 0.02). Data represent the mean ± S.E.M. of four separate experiments and are expressed as picomoles per 106 cells/group. , p < 0.05; , p < 0.01 compared with vehicle-treated group, respectively.

MMT Facilitates ROS Generation. Reduction of m is an index of mitochondrial dysfunction, and mitochondria are considered major sources of oxidative stress. When mitochondria are impaired, more ROS may be generated (Voehringer and Meyn, 2000). Enhanced generation of ROS was observed in PC-12 cells at 1 h after MMT treatment as measured by the ROS-detecting fluor DCF-DA (Fig. 3A). MMT treatment concentration dependently increased DCF-H fluorescence, the peroxidized product of DCF-DA. Maximal production of ROS by MMT treatment was 316.3% of vehicle control with an EC₅₀ for MMT treatment of 51.55 µM as determined by three-parameter logistic regression (r² = 0.93). Further support of ROS generation was confirmed by hydroethidine fluorescence measurements, which are relatively specific for superoxide radicals. Hydroethidine measurement of superoxide formation revealed a concentration-dependent increase in generation of ROS at 30 min post-treatment (Fig. 3B).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Concentration-dependent ROS generation in MMT-treated PC-12 cells. A, DCF-H, the fluorescent dye product of peroxidized DCF-DA, was measured fluorometrically in MMT-treated PC-12 cells at 1-h post-treatment and analyzed by three-parameter logistic regression (EC50 of 51.52 ± 1.93 µM; r2 = 0.94). B, hydroethidine, the fluorescent dye product of dihydroethidine, was measured by flow cytometer in MMT-treated PC-12 cells at various concentrations up to 30 min. Data represent the mean ± S.E.M. of three to nine experiments and are expressed as percentage of vehicle control. , p < 0.01 compared with vehicle-treated group.

MMT Activates Caspase-3 Activity in PC-12 Cells. Mitochondrial dysfunction and increased oxidative stress have been implicated in initiation of apoptosis in dopaminergic cells by treatment with various toxicants (Lotharius and O'Malley, 2000; Robertson and Orrenius, 2000; Kitazawa et al., 2001). Because MMT significantly increased intracellular ROS level within 1 h, other proapoptotic molecules may also be activated during the exposure period. We measured the activity of caspase-3, an effector cysteine-aspartate protease and one of the key proapoptotic molecules activated by various apoptotic stimuli, using caspase-3-specific fluorescent substrate Ac-DEVD-AMC after 0 to 200 µM MMT treatment in PC-12 cells. Caspase-3 was activated concentration dependently, and caspase-3 activity showed 6- and 15-fold increases from basal level with 150 and 200 µM MMT exposure for 1 h, respectively (Fig. 4A).

View larger version (26K): [in this window] [in a new window]   Fig. 4.   MMT-induced caspase-3 activation. A, PC-12 cells were exposed to MMT (0-200 µM) for 1 h, and caspase-3 activity was measured using a caspase-3-specific substrate, Ac-DEVD-AMC. B, PC-12 cells were pretreated with 1 mM Trolox or 5 mM NAC for 30 min or with 500 µM -MPT for 24 h before exposure to MMT. After 1-h MMT exposure then cytosolic fraction was collected, and caspase-3 activity was measured. All data represent the mean ± S.E.M. for three separate experiments in triplicate. , none; , +1 mM Trolox; , + 5 mM NAC; , +500 µM -MPT; , p < 0.01 compared with vehicle-treated group or , p < 0.05 compared with MMT alone-treated group.

MMT Induces Apoptosis in Dopamine-Producing PC-12 Cells. Chromatin condensation and DNA fragmentation are unique morphological changes during the terminal phases of apoptotic cell death. Treatment of PC-12 cells with MMT (200 µM) caused the formation of uncoiled DNA by qualitative assessment using acridine orange staining (Fig. 5A). Using a more sensitive ELISA-based method to measure DNA fragmentation, we observed approximately a 170% increase in DNA fragmentation with 200 µM MMT exposure within 1 h (Fig. 5B). Previously, we also demonstrated that caspase inhibitors Z-VAD-FMK and Z-DEVD-FMK effectively attenuated MMT-induced DNA fragmentation, thus caspase-3 plays an important role in apoptosis after MMT exposure (Anantharam et al., 2002).

View larger version (54K): [in this window] [in a new window]   Fig. 5.   MMT-induced DNA fragmentation in PC-12 cells. A, qualitative assessment of nuclear degradation in 200 µM MMT-treated PC-12 cells stained with acridine orange at 1 h. B, PC-12 cells were treated (30 min) with 1 mM Trolox before treatment with 200 µM MMT. DNA fragmentation was measured by DNA ELISA assay after 1 h of MMT treatment. Asterisks represent results of a Welch-corrected unpaired t test comparing either Trolox alone or Trolox + MMT with MMT alone. Data are expressed as percentage of vehicle-treated group (mean ± S.E.M.) of three to six experiments. , p < 0.05; , p < 0.01 compared with MMT-treated group; , p < 0.01 compared with vehicle-treated group.

Bcl-2 Attenuates MMT-Altered Loss of m. To better substantiate the evidence from pharmacological studies performed herein with respect to specific implication of mitochondrial dysfunction as a key factor in initiation of apoptotic cell death, we tested the effect of MMT on a transfected PC-12 cell line overexpressing the apoptotic control protein Bcl-2. Verification of the level of Bcl-2 expression in vector-only control (PC-12V4) and Bcl-2-overexpressed (PC-12HB2-3) cell lines was performed by Western blot (Fig. 6A), with verification of equal protein loading per gel lane by reprobe with anti-HSP60. To measure mitochondrial-specific effects, we used DiOC₆ to measure reduction of m after MMT treatment. Acute (1 h) MMT exposure concentration dependently decreased m, and the reduction was significant (p < 0.01) at 150 and 200 µM MMT in PC-12V4 cells (Fig. 6B). The reduction observed was 58.6 and 48.2% of vehicle-treated group after 150 and 200 µM MMT exposure for 1 h, respectively, whereas Bcl-2-overexpressed cells showed 106.8% at 150 µM MMT exposure, which completely attenuated the depolarization of m, and 72.2% at 200 µM MMT. Attenuation of m by Bcl-2 was significant (p < 0.05) at both concentrations of MMT.

View larger version (43K): [in this window] [in a new window]   Fig. 6.   Overexpression of Bcl-2 protects PC-12 cells against MMT-induced loss of m. A, overexpression of Bcl-2 in expression vector control (PC-12V4) and Bcl-2-enhanced expression positive (PC-12HB2-3) cells as verified by Western blot (top). Verification of equal protein loading per gel lane was verified by membrane reprobe for HSP60, a basic mitochondrial protein (bottom). B, PC-12V4 (vector control; ) and PC-12HB2-3 (Bcl-2 overexpressing; ) cells were exposed to 0 to 200 µM MMT for 1 h, and reduction of m was determined by flow cytometer using 40 nM DiOC6. Relative fluorescence intensity was measured and expressed as percentage of vehicle-treated group. Data represent the mean ± S.E.M. for two separate experiments in triplicate. , p < 0.05; , p < 0.01 compared with vehicle-treated group or between indicated treatment groups.

Bcl-2 Reduces MMT-Induced ROS Generation. To examine whether attenuation of MMT-induced depolarization of m by Bcl-2 was the result of blocked ROS generation, we measured ROS generation in PC-12V4 and PC-12HB2-3 cells after MMT treatment. It has previously been shown that MMT rapidly increases cellular ROS level within 1 h, and the peak ROS level is around 15 to 30 min after MMT treatment (Anantharam et al., 2002). As shown in Fig. 7, A and B, ROS generation increased after MMT exposure for 15 or 30 min in PC-12V4 cells. Conversely, Bcl-2-overexpressed PC-12HB2-3 cells showed reduced ROS generation, which is significantly (p < 0.01) less than the levels in PC-12V4 cells. Thus, Bcl-2 protein overexpression seems to attenuate MMT-induced ROS generation.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Overexpression of Bcl-2 significantly reduces MMT-mediated ROS generation. PC-12V4 (vector control; ) and PC-12HB2-3 (Bcl-2 overexpressing; ) cells were exposed to 0 to 200 µM MMT, and fluorescence intensity of dihydroethidine was measured by flow cytometer at 15 min (A) and 30 min (B) after MMT treatment. Data represent the mean ± S.E.M. of three experiments in triplicate and are expressed as percentage of vehicle control. , p < 0.05; , p < 0.01 compared with vehicle-treated group or between indicated treatment groups.

Bcl-2 Overexpression Protects against Caspase-3 Activation and DNA Fragmentation. MMT-mediated mitochondrial dysfunction, as measured by the reduction of m, was effectively attenuated by Bcl-2 overexpression, suggesting that downstream cell death processes could be blocked only if mitochondria regulate MMT-induced apoptotic cell death. As shown in Fig. 8A, PC-12V4 cells showed 18- and 23-fold increase in caspase-3 activity after 150 and 200 µM MMT exposure, respectively. As we expected, caspase-3 activity in PC-12HB2-3 cells was significantly (p < 0.01) blocked after 1-h MMT (150 µM) exposure, and increased caspase-3 activity was only observed at 200 µM MMT exposure.

View larger version (18K): [in this window] [in a new window]   Fig. 8.   Overexpression of Bcl-2 protects PC-12 cells against MMT-induced caspase-3 activation and nuclear DNA fragmentation. A, PC-12V4 (vector control; ) and PC-12HB2-3 (Bcl-2 overexpressing; ) cells were exposed to MMT (0-200 µM) for 1 h, and caspase-3 activity was measured. Caspase-3 activity was expressed as relative fluorescence units (FU) per milligram of protein per hour of incubation with caspase-3 substrate at 37°C. All data represent the mean ± S.E.M. for three separate experiments in triplicate. *, p < 0.05 or **, p < 0.01 compared with vehicle-treated group or between indicated treatment groups. B, PC-12V4 and PC-12HB2-3 cells were exposed to MMT (0-200 µM) for 1 h, and DNA fragmentation was measured by ELISA method. Data represent the mean ± S.E.M. for two separate experiments in duplicate. *, p < 0.05 or **p < 0.01 compared with vehicle-treated group or between indicated treatment groups.

	Discussion

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Health effects regarding the use of MMT as a tetraethyl lead replacement in automotive fuels have been studied predominantly from the aspect of manganese particulate combustion products. However, data presented herein delineate a health risk potential associated with effects of the parent chemical. Accidental exposures through spills or unintended uses of MMT-amended gasoline provides a routine human exposure route, which, noting both the demonstrated effects of manganese on dopaminergic neurochemical systems (Olanow et al., 1996; Rodruigez et al., 1998) and long blood plasma half-life of MMT (Zheng et al., 2000), may potentially play a role in environmentally mediated, geriatric onset PD. Recent epidemiological and case-control studies support the role of environmental exposure to metals and other organic toxicants such as pesticides in idiopathic PD (Gorrel et al., 1999; Tanner et al., 1999).

One of the earliest cellular responses after MMT exposure is ROS generation. Oxidative stress has also been shown to mediate manganese-induced apoptosis in several in vitro models such as PC-12 cells and HeLa cells (Desole et al., 1997b; Oubrahim et al., 2001) and in vivo in manganese-induced neurotoxicity of the rat (Desole et al., 1997a), which is relevant within the context of combustion products of MMT-amended fuels. Because MMT seems to exert cytotoxic effects also as an organic complex of manganese, the potential danger of MMT-amended fuels is possibly enhanced by both precombustion (MMT) and postcombustion (manganese) product exposures (Garrison et al., 1995). In this study, we have shown MMT-induced activation of apoptotic signals in PC-12 cells and have further elucidated the mechanisms of MMT-mediated cell death, which could have implications for effects of MMT on dopaminergic neuronal systems.

The results of the present study clearly demonstrate that the cytotoxic ability of MMT in in vitro systems resides in the capacity of MMT to kill cells by a mitochondria-mediated apoptotic mechanism and oxidative stress. One potential additional mechanism for MMT-induced oxidative stress was suggested by the apparent ability of MMT to produce DOPAC formation accompanied with intracellular dopamine depletion. It has been demonstrated previously in a number of studies (Spina and Cohen, 1989; Fabre et al., 1999) that DOPAC metabolism generates H₂O₂ as a by-product of the reaction, either alone or enhanced in the presence of metals, which could also add to ROS generation observed herein.

Previous studies have demonstrated that certain environmental cyclodiene neurtoxicants such as dieldrin (Kitazawa et al., 2001) and heptachlor epoxide (Kirby et al., 2001) evoke dopamine release, which could have deleterious effects toward generation of ROS and activation of apoptotic pathways as a result of cytosolic pooling of recycled dopamine. Apoptosis resulting from cytosolic pooling of recycled dopamine can be attenuated by blocking expression of the presynaptic dopamine transporter (Simantov et al., 1996). However, the results of the present work suggest that MMT-induced dopamine degradation may be an important event in dopaminergic toxicity. Striatal dopamine depletion has been observed in mice treated with MMT (Gianutsos and Murray, 1982) and may occur in vivo by this mechanism involving degradation of dopamine. Of additional note, recent work by Lotharius and O'Malley (2000) demonstrates that MPP⁺ exerts neurotoxic effects not only by complex I inhibition but also by redistributing dopamine from the vesicular pool to the cytoplasm, which fosters conversion of dopamine to various neurotoxic quinones. However, the authors did not find an MPP⁺-mediated reduction in m, in contrast to their results with rotenone, and suggest the existence of a peripheral source of ROS generation leading to catecholamine quinone production. In this respect, the effects of MMT on m seem to better resemble the rotenone Parkinsonism model (Betarbet et al., 2000) and may potentially combine the mitochondrial impairment aspect of this model with the catecholamine quinone formation aspect of the MPP⁺ model.

MMT cytotoxicity seems to be mediated by oxidative stress produced by both mitochondrial impairment and alteration of dopamine catabolism. Previous studies by Autissier et al. (1977a,b) demonstrated direct effects of MMT on mitochondrial complex I, and determined that the Mn²⁺ component of MMT is specifically responsible for altering the electronic configuration of the carbonyl groups to promote association with complex I. Additionally, MMT not only interferes with NAD⁺-linked substrate energy transfer but also interferes with electron donation to ubiquinone, which results in a decrease in oxidative phosphorylation (Autissier et al., 1977a). In our studies, measurement of the increase in activity of proapoptotic messengers (caspase-3) and expression of cytotoxic indices (increased DNA fragmentation) can be collectively inhibited by various protective agents such as Trolox (antioxidant) or -MPT (tyrosine hydroxylase inhibitor). Deprenyl (monoamine oxidase-B inhibitor) protected cells from DNA fragmentation, but did not have a measurable effect on reduction of caspase-3 activity, which suggests that MMT-induced DNA fragmentation can occur by a caspase-3-independent pathway(s) as observed by other researchers (Volbracht et al., 2001). Regardless, the majority of our results implicate the mitochondria as either the source of or target of ROS and link the process of apoptosis with early events directed at mitochondrial damage. The protective effects of tyrosine hydroxylase inhibition additionally implicate dopamine or dopamine by-products as contributing components of apoptotic initiation and may partially help to explain the effects of MMT in vivo on dopaminergic systems (Gianutsos and Murray, 1982). Additionally, previous studies have demonstrated that inorganic manganese exposure produces selective neurotoxic effects on dopaminergic systems, including the nigrostriatal tract (Olanow et al., 1996; Rodruigez et al., 1998).

We further confirmed the mitochondrial impairment aspect of MMT cytotoxicity by constructing PC-12-derived cell lines specifically tailored to overexpress the apoptotic control protein Bcl-2. In the normal state, phosphorylated Bcl-2 in the mitochondrion forms stable heterodimers with proapoptotic control proteins in the same gene family (Robertson and Orrenius, 2000; Adams and Cory, 2001). Bcl-2 heterodimeric complexes have the ability to inhibit apoptotic processes by direct interference with proapoptotic messengers (e.g., Bax and Bak), to prevent opening of the mitochondrial permeability transition pore, and by unknown mechanisms foster increased concentrations of reduced glutathione in the nuclear envelope and augmentation of the NADPH energy pool in mitochondria (Voehringer and Meyn, 2000). The latter two effects also possibly have roles in both down-regulation of apoptotic pathways and increased transcription of antiapoptotic/homeostatic genes dependent upon the redox status of the cell, a perspective based on several lines of evidence and advocated by Voehringer and Meyn (2000). Indeed, in the present study, stabilization of intracellular redox status by application of antioxidants seems to compliment hypotheses of Bcl-2 function regarding increased expression or activity of ROS-protective mechanisms.

Dephosphorylation of Bcl-2 tends to favor release of Bax and Bak to form homodimers and/or Bax/Bak heterodimers. These Bax and Bak formations promote cytochrome c release and mitochondrial transition pore opening, the latter of which results in loss of m (Adams and Cory, 2001). Stabilization of mitochondrial transition pore closed state in Bcl-2-overexpressed cells was verified by lack of reduction in m after MMT exposure, whereas a loss of m was observed in vector control cells treated with MMT. We also previously reported that MMT-induced caspase-3 activation is mediated by cytochrome c release from mitochondria (Anantharam et al., 2002), which follows currently understood toxicant-induced apoptosis models (Tsujimoto, 1998; Robertson and Orrenius, 2000). After cytochrome c release, apoptosis protease-activating factor-1 release from dephosphorylated Bcl-2 and subsequent apoptosis protease-activating factor-1 dimerization to cytosolic cytochrome c promote cleavage and activation of procaspase-9, an important initiator caspase signaling downstream apoptotic events. These caspase-9-dependent events include, but perhaps may not be limited to, cleavage and activation of procaspase-3 and procaspase-7. Caspase-3 in particular is known to be a critical effector caspase (Abu-Qare and Abou-Donia, 2001; Adams and Cory, 2001), which activates a host of downstream proapoptotic effectors (e.g., protein kinase C- and DNA-dependent protein kinase), inactivates a variety of downstream antiapoptotic effectors [e.g., poly(ADP-ribose) polymerase and inhibitor of caspase-activated deoxyribonuclease], and cleaves cytoskeletal structural proteins (e.g., spectrin, actin, and lamin). Overexpression of Bcl-2 in its normal phosphorylated state provides an abundant pool for sequestration of mitochondrial proapoptotic messengers and further underscores the results of the pharmacological studies reported herein, which suggest that initiation of apoptosis by MMT occurs through mitochondrial dysfunction.

In conclusion, the results presented herein delineate that oxidative stress plays an important role in mitochondrial-mediated apoptotic cell death in cultured dopamine-producing cells after exposure to MMT. Forthcoming studies conducted in this laboratory will attempt to demonstrate whether nigrostriatal neurons are selectively susceptible to MMT-mediated neurotoxicity.