Methylmercury Impairs Canonical Dopamine Metabolism in Rat Undifferentiated Pheochromocytoma (PC12) Cells by Indirect Inhibition of Aldehyde Dehydrogenase

Toxicological Sciences, Apr 2015

The environmental neurotoxicant methylmercury (MeHg) disrupts dopamine (DA) neurochemical homeostasis by stimulating DA synthesis and release. Evidence also suggests that DA metabolism is independently impaired. The present investigation was designed to characterize the DA metabolomic profile induced by MeHg, and examine potential mechanisms by which MeHg inhibits the DA metabolic enzyme aldehyde dehydrogenase (ALDH) in rat undifferentiated PC12 cells. MeHg decreases the intracellular concentration of 3,4-dihydroxyphenylacetic acid (DOPAC). This is associated with a concomitant increase in intracellular concentrations of the intermediate metabolite 3,4-dihydroxyphenylaldehyde (DOPAL) and the reduced metabolic product 3,4-dihydroxyethanol. This metabolomic profile is consistent with inhibition of ALDH, which catalyzes oxidation of DOPAL to DOPAC. MeHg does not directly impair ALDH enzymatic activity, however MeHg depletes cytosolic levels of the ALDH cofactor NAD+, which could contribute to impaired ALDH activity following exposure to MeHg. The observation that MeHg shunts DA metabolism along an alternative metabolic pathway and leads to the accumulation of DOPAL, a reactive species associated with protein and DNA damage, as well as cell death, is of significant consequence. As a specific metabolite of DA, the observed accumulation of DOPAL provides evidence for a specific mechanism by which DA neurons may be selectively vulnerable to MeHg.

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Methylmercury Impairs Canonical Dopamine Metabolism in Rat Undifferentiated Pheochromocytoma (PC12) Cells by Indirect Inhibition of Aldehyde Dehydrogenase

TOXICOLOGICAL SCIENCES Methylmercury Impairs Canonical Dopamine Metabolism in Rat Undifferentiated Pheochromocytoma (PC12) Cells by Indirect Inhibition of Aldehyde Dehydrogenase Chelsea T. Tiernan 1 3 4 Ethan A. Edwin 0 1 3 Hae-Young Hawong 1 2 3 6 Mo´ nica Rı´os-Cabanillas 0 1 3 John L. Goudreau 0 1 3 4 5 William D. Atchison 0 1 3 4 Keith J. Lookingland 0 1 3 4 0 Department of Pharmacology and Toxicology 1 Michigan State University , East Lansing, Michigan 48824 , USA 2 Department of Biochemistry 3 Molecular Biology 4 Neuroscience Program 5 Department of Neurology and Ophthalmology 6 College of Osteopathic Medicine The environmental neurotoxicant methylmercury (MeHg) disrupts dopamine (DA) neurochemical homeostasis by stimulating DA synthesis and release. Evidence also suggests that DA metabolism is independently impaired. The present investigation was designed to characterize the DA metabolomic profile induced by MeHg, and examine potential mechanisms by which MeHg inhibits the DA metabolic enzyme aldehyde dehydrogenase (ALDH) in rat undifferentiated PC12 cells. MeHg decreases the intracellular concentration of 3,4-dihydroxyphenylacetic acid (DOPAC). This is associated with a concomitant increase in intracellular concentrations of the intermediate metabolite 3,4-dihydroxyphenylaldehyde (DOPAL) and the reduced metabolic product 3,4-dihydroxyethanol. This metabolomic profile is consistent with inhibition of ALDH, which catalyzes oxidation of DOPAL to DOPAC. MeHg does not directly impair ALDH enzymatic activity, however MeHg depletes cytosolic levels of the ALDH cofactor NADþ, which could contribute to impaired ALDH activity following exposure to MeHg. The observation that MeHg shunts DA metabolism along an alternative metabolic pathway and leads to the accumulation of DOPAL, a reactive species associated with protein and DNA damage, as well as cell death, is of significant consequence. As a specific metabolite of DA, the observed accumulation of DOPAL provides evidence for a specific mechanism by which DA neurons may be selectively vulnerable to MeHg. VC The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: methylmercury-induced neurotoxicity; dopamine metabolism; PC12 cell; aldehyde dehydrogenase - The neurotransmitter dopamine (DA) is metabolized in a 2-step process. First, monoamine oxidase (MAO) catalyzes the oxidative deamination of DA to 3,4-dihydroxyphenylaldehyde (DOPAL). Second, this reactive intermediate metabolite either undergoes further oxidation mediated by aldehyde dehydrogenase (ALDH) to form the carboxylic acid 3,4-dihydroxyphenylacetic acid (DOPAC), or it is reduced by aldehyde/aldose reductase (AR) to form the alcohol 3,4-dihydroxyethanol (DOPET) (Meiser et al., 2013) . These “oxidative” and “reductive” pathways of DA metabolism are illustrated in Figure 1. DA is predominately degraded along the “oxidative” metabolic pathway due to the absence of a b-hydroxyl group. This is in contrast to the other catecholamines such as norepinephrine and epinephrine which contain a b-hydroxyl group, and are preferentially degraded along the “reductive” pathway (Turner et al., 1974) . Tight regulation of DA metabolism is necessary because both cytosolic DA and DOPAL can be cytotoxic (Graham et al., 1978; Rees et al., 2009) . Cytosolic DA undergoes autooxidation in the presence of molecular oxygen to form either a quinone or semiquinone (Le´vay and Bodell, 1993) . These reactive species can covalently bind cellular proteins and DNA (Graham et al., 1978) . Similarly, DOPAL forms adducts with amino acids, and cross-links proteins and DNA (Nair et al., 1986; Rees et al., 2009) . Subsequent biological effects of excess DA and its aldehyde adducts could contribute to inactivation of enzymes, depletion of antioxidant systems, or altered signal transduction, gene expression, and DNA repair (Marchitti et al., 2007; Stokes et al., 1999) . Indeed, the specific vulnerability and death of DA neurons in Parkinson disease is hypothesized to involve the accumulation of these toxic compounds selectively in DA neurons (Burke et al., 2003) . Methylmercury (MeHg) is a potent environmental neurotoxicant known to alter DA neurochemical homeostasis (Dreiem et al., 2009; Faro et al., 2002) . DA release is stimulated following exposure to MeHg both in vitro and in vivo (Faro et al., 2002; Kalisch and Racz, 1996) . Previous work from our laboratory using undifferentiated rat pheochromocytoma (PC12) cells demonstrates that MeHg-induced DA release is potentiated by activation of tyrosine hydroxylase, the rate limiting enzyme in DA synthesis (Tiernan et al., 2013). In contrast, while MeHg stimulates DA synthesis and release, the toxicant impairs canonical DA metabolism. Concentrations of DOPAC are reduced in striatal tissue slices and synaptosomes following exposure to MeHg (Bemis and Seegal, 1999; Dreiem et al., 2009) . Therefore, the observation that MeHg increases DA concentrations while decreasing DOPAC concentrations suggests that MeHg targets some process intrinsic to DA metabolism, independent of its effects on DA synthesis and release. MeHg-induced inhibition of oxidative DA metabolism could result from inhibition at the level of either MAO or ALDH. MeHg has a high affinity for sulfhydryl groups (Hughes, 1957) , and thus could bind directly to cysteine residues present in the active site of either MAO (Hubalek et al., 2003) or ALDH (Hempel et al., 1985) to impair enzymatic activity. Additionally, both MAO and ALDH are mitochondrial-associated enzymes (Marchitti et al., 2007; Schnaitman et al., 1967) . MeHg inhibits mitochondrial respiration (Sone et al., 1977), and as such could indirectly compromise the enzymatic activity of either MAO or ALDH. Impaired MAO or ALDH activity would be indicated by changes in the metabolomic profile of DA such that alterations in the ratios of DOPAC, DOPAL, and DOPET would denote the level of enzymatic inhibition. For example, inhibition of MAO activity would be reflected as a decrease in both DOPAL and DOPAC, with a concomitant increase in DOPET, whereas inhibition of ALDH activity would be associated with a decrease in DOPAC and an increase in DOPAL and DOPET. The goals of the present study were to (1) characterize the DA metabolomic profile following exposure to MeHg in undifferentiated PC12 cells and (2) examine the specific contributions of direct and indirect ALDH inhibition to MeHgimpaired DA metabolism. To this end, the effects of MeHg on DA metabolism, ALDH activity, and mitochondrial function were compared with those of the reversible ALDH2 inhibitor daidzin (Keung and Vallee, 1993) and the reversible mitochondrial electron transport chain (ETC) Complex I inhibitor rotenone (Oberg, 1961). Because MeHg has multiple, complex targets, an in vitro model was used to isolate discrete components of DA homeostasis altered by this neurotoxicant. Undifferentiated PC12 cells contain all of the enzymes necessary for DA metabolism (Greene and Rein, 1977) and the effects of MeHg have been previously described in this system (Shafer and Atchison, 1991) . MATERIALS AND METHODS Chemicals and solutions. Cell culture supplies, including RPMI-1640 Medium, horse serum, trypsin, and penicillin-streptomycin, were purchased from GIBCO BRL (Grand Island, New York). Hyclone fetal bovine serum was purchased from Thermo Scientific (Logan, Utah). Methyl mercuric chloride (MeHg) was purchased from ICN Biochemicals, Inc (Aurora, Ohio). Unless otherwise stated, all remaining chemicals were purchased from SigmaAldrich (St Louis, Missouri). The standard physiological saline used for extracellular solution was HEPES-buffered saline (HBS), which contained (mM): 150 NaCl, 5 KCl, 2.4 CaCl2, 1.6 MgSO4, 20 HEPES, and 20 d-glucose (pH 7.3). MeHg was prepared as a 10 mM stock solution in distilled water. Daidzin and rotenone were dissolved in 100% DMSO as 10 mM stock solutions and then diluted so the final concentration of DMSO was less than 0.05% (vol/vol). All compounds were diluted to working concentrations in HBS on the day of each experiment. Culture of PC12 cells. PC12 cells (Gift of Dr M. L. Contreras) were grown in RPMI-1640 Medium supplemented with 10% (vol/vol) horse serum, 2.5% (vol/vol) fetal bovine serum, and 1% (vol/vol) penicillin-streptomycin (pH 7.3). Cultures were maintained in either 25-cm2 or 75-cm2 T-flasks in a humidified environment containing 5% CO2 at 37 C. Culture medium was changed every 2–3 days. Every 4–5 days PC12 cultures were detached from the flasks with 0.25% (vol/vol) trypsin and subcultured at a density of 3 105cells/ml for a 5-day culture or 4 105cells/ml for a 4-day culture. All cultures were maintained at 80%–90% confluence at the time of subculture. To maintain consistency from experiment to experiment, cells were used between passages 16–19 from our receipt. Experimental conditions were repeated in biological triplicate and experiments were replicated at least 3 times from separate cultures to minimize the risk of culturespecific confound. PC12 cell treatment. Undifferentiated PC12 cells were seeded in 6-well plates coated with poly-D-lysine at a density of 6 105 cells/ml 48 h prior treatment. Culture medium was aspirated and replaced with HBS or HBS containing 1, 2, or 5 lM MeHg and incubated at 37 C/5% CO2 for 15, 30, 60, or 120 min (Tiernan et al., 2013) . In some experiments, the effects of 2 lM MeHg were compared to those of either 20 lM daidzin for 60 min or 50 nM rotenone for 15 min. Concentrations and time points for these comparative chemicals were selected based on reports in the literature demonstrating changes in the DA metabolomic profile (Keung and Vallee, 1993; Lamensdorf et al., 2000b) . Measurements of intracellular neurochemistry. At experiment termination, 6-well plates were centrifuged at 300 g for 5 min at 4 C, and then treatment medium was aspirated. Cells were rinsed once with 1 ml ice-cold phosphate-buffered saline, harvested, and pelleted by centrifugation at 12 000 g for 5 min at 4 C. After centrifugation, the supernatant was removed and replaced with 100 ll of ice-cold tissue buffer. The content of DA metabolites in the supernatant was determined using high-pressure liquid chromatography coupled with electrochemical detection (HPLCED) using a Water 515 HPLC pump (Waters Corp, Milford, Massachusetts) and an ESA Coulochem 5100 A electrochemical detector with an oxidation potential of þ0.4 V. Neurochemical content was quantified by comparing peak height of each sample to peak heights of standards and normalized to mg protein as determined by the bicinchoninic acid protein assay (Sigma). Synthesis of DOPAL standard. To quantify DOPAL concentrations following experimental manipulations, a DOPAL standard was synthesized. DA (Sigma) was dissolved in phosphate buffer (140 mM NaCl, 8.1 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.5) to a final concentration of 1 mM. Phosphate-buffered DA (750 ll) was incubated in a 12 75 mm culture tubes at 37 C for 15 min; 10 U of MAO (BD Bioscience, San Jose, California) was added to the culture tube to start the reaction, and the tube was incubated at 37 C for 45 min. The reaction was terminated by addition of 450 ll icecold tissue buffer. Samples were centrifuged at 12 000 g for 10 min, and supernatant was removed and placed into a fresh tube for HPLC-ED analysis of DA and DOPAL. Concentrations of DA were compared with 1 ng standards of DA, and amount of DA lost at 45 min. Supplementary Figure 1 illustrates the time course of DA conversion to DOPAL. ALDH enzyme kinetics assay. Following treatment, 6-well plates were centrifuged at 300 g for 5 min at 4 C. Treatment medium was aspirated and replaced with 150 ll homogenization buffer (0.1 M Tris-HCl, 10 mM dithiothreitol, 20% glycerol [vol/vol], and 1% Triton-X [vol/vol]). Cells were scraped and then pelleted by centrifugation at 13 000 g for 8 min at 4 C. Protein concentration of supernatant was determined using the bicinchoninic protein assay. ALDH enzyme activity was determined spectrophotometrically by monitoring the reductive reaction of NADþ to NADH at 340 nm using the Tecan Infinite M1000 Pro microplate reader and Magellan data analysis software (Tecan Systems, Inc, San Jose, California). Assays were carried out in 96-well plates containing 100 mM Tris-HCl buffer (pH 8.0) at 30 C. Nicotinamide adenosine dinucleotide hydrate (NADþ, 2 mM) and 100 mg PC12 cell lysate were added to the buffer. In some experiments, 2 lM MeHg or 20 lM daidzin was added to the assay wells. To start the reaction, 10 mM propionaldehyde was added. Accumulation of NADH was recorded every 30 s for 10 min. Substrate blanks (NADþ þ lysate) were run simultaneously and results were corrected for blank reactions. ALDH reaction rates were calculated as mmole NADH per min per mg protein. Measurements of mitochondrial bioenergetics. Mitochondrial respiration was measured as the rate of oxygen consumption (OCR) using the XF24 Extracellular Flux Analyzer (Seahorse Bioscience, Billerica, Massachusetts). Cells plated in a modified 24-well plate were fit with a biosensory cartridge containing 24 fluorophores (1 per well) sensitive to molecular oxygen. OCR was measured within a transient microchamber created in each of the 24 wells when the biosensor cartridge was lowered 200 lm above the cells. Each cartridge was also equipped with 4 reagent delivery ports per well for dispensing testing agents at specified times during an assay. In the present set of experiments, injection ports were loaded with oligomycin A, carbonyl cyanide-ptrifluoromethoxyphenylhydrazone (FCCP), and antimycin A. Basal respiration was measured first and then chemicals were injected sequentially to measure OCR following inhibition of the ATP synthase (oligomycin A), uncoupling of electron transporter and ATP synthesis (FCCP), and inhibition of electron transport (antimycin A). OCR measured under these conditions was used to calculate 3 parameters of mitochondrial respiration: basal respiration, ATP turnover, and maximum or spare respiration. Undifferentiated PC12 cells were seeded in XF24-well cell culture microplates coated with poly-D-lysine at a density of 7 105 cells in 100ll RPMI culture medium. The cell culture plate was incubated at 37 C/5% CO2 for 1 h before 150 ll of additional culture medium was added carefully to each well and cells were incubated at 37 C/5% CO2 for 48 h. On the day of each experiment, culture medium was aspirated and replaced with HBS or HBS containing either 2 lM MeHg or 50 nM rotenone, and cells were incubated at 37 C/5% CO2 for 15–60 min. Following treatment, cells were rinsed with 1 ml warmed XF24 assay medium (RPMI-1640, pH 7.3, supplemented with 2.5 mM d-glucose), and then XF24 assay medium was brought to a final volume of 675 ll. Cells were incubated at 37 C/no CO2 for 1 h to allow medium temperature and pH to equilibrate before the first measurements were made. For the XF assay, the cell culture plate was placed in the calibrated XF24 Flux Analyzer. Baseline OCR (nM/min) was measured 3 times. Between each rate measurement, assay medium was gently mixed by the XF24 Analyzer for 3–5 min to restore normal oxygen tension in the microchamber surrounding the cells. Following baseline measurement, injection ports were sequentially injected to reach test compound final concentrations and the medium was mixed for 3–5 min before OCR was measured 3 times, as described for baseline measurements. NADH/NAD1 quantification. Undifferentiated PC12 cells were plated and treated with 2 lM MeHg as described above. To terminate treatment, cell plates were centrifuged at 300 g for 5 min and treatment medium was aspirated. Cells were washed once with ice-cold phosphate-buffered saline, and then pelleted by centrifugation at 10 000 g for 5 min at 4 C. NADH/NAD was extracted using 400 ml NADH/NAD Extraction Buffer (Sigma) by 2 freeze/thaw cycles (20 min on dry ice followed by 10 min at room temperature). Samples were homogenized and centrifuged for 10 min at 13 000 g to remove insoluble material. To quantify NADþ, samples were measured as described below. For NADH measurements, 200 ml was incubated at 60 C for 30 min before measurement. A 96-well plate was loaded with 50 ml sample and 100 ml Master Reaction Mix (NAD Cycling Buffer þ NAD Cycling Enzyme; Sigma). Samples were incubated for 5 min to convert NAD to NADH, and then 10 ml NADH Developer was added to each well. Samples were incubated for 1 h at room temperature. Absorbance at 450 nm was measured using the Tecan Infinite M1000 Pro microplate reader with Magellan data analysis software (Tecan Systems, Inc). Average absorbance was compared with NADH standards to calculate the concentration of total NADþ, total NADH, and the ratio of NADþ: NADH. Statistical analysis. SigmaPlot software version 12.0 (SysStat Software, Inc, Point Richmond, California) was used to make statistical comparisons among groups using unpaired t test, 1way ANOVA, or non-parametric alternatives as appropriate. If a significant difference was detected, post hoc between-group comparisons were performed using Tukey’s test. Statistical significance was set at P 0.05. RESULTS MeHg Impairs Canonical DA Metabolism and Shunts DA along an Alternative Metabolic Pathway Concentration-response and time-course effects of MeHg on intracellular DA and DOPAC concentrations in undifferentiated PC12 cells are presented in Figure 2. At lower concentrations (1 lM), MeHg induces variable changes in intracellular DA and DOPAC, whereas at higher concentrations (2–5 lM) there is a significant, reproducible increase in DA with a concomitant decrease in DOPAC (Figs. 2 and 3A and 3B). This observation suggests that MeHg impairs canonical DA metabolism by inhibiting some process essential to DOPAC formation. To evaluate further the mechanisms of impaired DA metabolism, the 2 lM MeHg concentration at the 60 min time point was selected. At this concentration and time, any observed changes in the DA metabolomic profile result from MeHg itself and not overt cytotoxicity (Tiernan et al., 2013) . The observed decrease in intracellular DOPAC is in direct contrast to the effect of MeHg on the intermediate DA metabolite DOPAL and the reduced metabolic product DOPET. Both of these are increased following exposure to 2 lM MeHg for 60 min (Figs. 3C and 3D). Comparison of the DA metabolomic profile in control and MeHg-treated PC12 cells revealed that treatment with MeHg shifts the percentage distribution of DA metabolites (Fig. 4). Under basal conditions, DOPAC comprises 56% of total metabolites, while DOPAL and DOPET account for 31% and 13%, respectively. Following exposure to MeHg, the concentration of DOPAC decreases to account for only 14.5% of the total metabolites, whereas DOPAL and DOPET concentrations increase to account for 53.5% and 32%, respectively. Additionally, MeHg significantly increases the overall total concentration of DA metabolites (ie, DOPAC, DOPAL, and DOPET) from 257.9 6 31.4 to 398.1 6 40.2 ng/mg protein (P < 0.05, HBS vs MeHg). These data together suggest that MeHg impairs DOPAC formation at the level of the DA metabolic enzyme ALDH, as inhibition of MAO activity would have decreased both DOPAL and DOPAC formation. Based on these data, additional experiments were designed to determine whether MeHg directly or indirectly impairs ALDH activity. Comparison of the Effects of MeHg, Daidzin, and Rotenone on DA Metabolism MeHg could impair ALDH activity directly by interacting with sulfhydryl groups present in the active site of the enzyme (Hempel et al., 1985) . It could also indirectly decrease ALDH activity by decreasing availability of the ALDH cofactor, NADþ, which is provided by Complex I of the mitochondrial ETC (Lamensdorf et al., 2000a,b) . In order to discriminate direct versus indirect effects of MeHg, the DA metabolomic profile induced by MeHg was compared with that produced by daidzin, a selective ALDH2 inhibitor (Keung and Vallee, 1993) , or rotenone, which inhibits Complex I of the mitochondrial ETC (Complex I of the mitochondrial ETC; Oberg, 1961) . The comparative effects of treatment with 2 lM MeHg, 20 lM daidzin, or 50 nM rotenone on concentrations of DA metabolites are presented in Figure 5. All 3 compounds reduced the concentration of intracellular DOPAC by approximately 60%. Only MeHg and daidzin increased the concentration of intracellular DOPET, but not to the same extent; MeHg increased DOPET by over 300%, whereas daidzin only increased DOPET by approximately 200%. ALDH Activity Is Not Directly Inhibited by MeHg The comparative effects of MeHg and daidzin on ALDH enzymatic activity are presented in Figure 6. ALDH activity was measured as NADH production under 2 conditions, either following a 60 min pretreatment or in the continued presence of MeHg or daidzin (20 lM). Following pretreatment, neither daidzin nor MeHg altered NADH production (Fig. 6A), whereas when ALDH activity was measured in the presence of each chemical, daidzin decreased NADH production (Fig. 6B). MeHg had no effect on NADH production under either experimental condition, indicating no direct effect on ALDH enzymatic activity. MeHg Impairs Mitochondrial Function and Decreases Availability of the ALDH Cofactor NAD1 A possible indirect effect of MeHg on ALDH activity was investigated by measuring mitochondrial function and intracellular production of the ALDH cofactor NADþ. Mitochondrial respiration was measured either following MeHg or rotenone (50 nM for 15 min) (Fig. 7). Following pretreatment with each toxicant, OCR was measured under basal conditions and then following successive treatments with 1 lM oligomycin A, 2 lM FCCP, and 1 lM antimycin A to assess basal respiration, ATP generation, maximum respiration, and non-mitochondrial respiration, respectively. The quantification of these effects revealed that MeHg attenuates basal and spare respiration, as well as ATP generation, whereas rotenone reduces only spare respiratory capacity without altering basal respiration or ATP generation (Fig. 8). Complex I of the mitochondrial ETC accepts electrons from NADH thereby forming NADþ as a byproduct. ALDH uses NADþ provided by Complex I (Lamensdorf et al., 2000b) , and MeHgimpaired mitochondrial respiration could detrimentally affect the production of NADþ. As such, we quantified the amount of the cofactor in undifferentiated PC12 cells treated with MeHg. As illustrated in Figure 9, MeHg not only decreased the amount of NADþ as anticipated, but also decreased NADH to a similar extent, such that there was no change in the NADþ/NADH ratio. DISCUSSION The present study provides evidence that MeHg impairs DA metabolism in undifferentiated PC12 cells through indirect inhibition of ALDH activity. These data are consistent with the following conclusions: (1) MeHg impairs conventional DA metabolism and shunts DA along an alternative reductive metabolic pathway, (2) MeHg does not directly impair ALDH activity, (3) mitochondrial dysfunction induced by MeHg decreases basal respiration, as well as ATP generation and spare respiratory capacity, and (4) MeHg-induced mitochondrial dysfunction decreases availability of the oxidized enzyme cofactor NADþ. The DA Metabolomic Profile Induced by MeHg MeHg inhibits DA metabolism. Previous reports demonstrate a concentration-dependent decrease in intracellular DOPAC in the striatum (Bemis and Seegal, 1999; Dreiem et al., 2009) , and decreased striatal release of DOPAC (Faro et al., 2002). Results from the present study support and extend these findings in dopaminergic undifferentiated PC12 cells. Intracellular DOPAC was significantly decreased following exposure to MeHg. This effect was observed at a range of concentrations (1–5 lM), and was sustained for the duration of evaluated endpoints (15–120 min). MeHg-induced decrease in intracellular DOPAC was accompanied by an increase in intracellular DA, suggesting that MeHg targets DA biosynthesis and degradation pathways by distinct, independent mechanisms. Because 2 lM MeHg significantly impaired DOPAC formation by 60 min while also stimulating DA synthesis, this concentration and time point were selected for further analysis of metabolic mechanisms. Previous work from our laboratory has demonstrated that at this concentration and time point, in undifferentiated PC12 cells, MeHg stimulates DA synthesis and release without inducing cell death (Tiernan et al., 2013) . Furthermore, at similar concentrations and time points, MeHg alters cellular and biochemical processes in a variety of cell types, including both astrocytes and neurons. For example, 3 lM MeHg stimulates adenosine triphosphate release from astrocytes in culture within 1 h of exposure, which stimulates production of the proinflammatory cytokine interleukin-6 (Noguchi et al., 2013) . Finally, in comparison to historical MeHg exposure in humans, the concentration evaluated presently is 1 order of magnitude lower than mean blood concentrations measured in Iraqi adults following acute poisoning in 1972 (Bakir et al., 1973) . MeHg-impaired DOPAC formation was accompanied by a significant and proportional increase in intracellular concentrations of DOPAL and DOPET. These data suggest that MeHg impairs DA metabolism by inhibiting ALDH, which oxidizes the toxic metabolite DOPAL to the inactive metabolite DOPAC. MAO appears to remain functional because the concentration of DOPAL does not decrease, indeed it increases. Likewise, results indicate that MeHg does not stimulate AR activity. Stimulation of AR would result in a profound increase in DOPET accumulation without a concomitant increase in DOPAL. While DOPET accumulation was moderately enhanced, this was expected as inhibition of the “oxidative pathway” has been previously demonstrated to increase DOPET formation (Lamensdorf et al., 2000b) . Inhibition of ALDH accounts for the observed neurochemical profile. DOPAL accumulates and its metabolism shifts to the lower-affinity “reductive pathway” mediated by AR, which enhances DOPET accumulation (Lamensdorf et al., 2000b; Turner et al., 1974) . The conclusion that MeHg inhibits ALDH is novel, however somewhat inconsistent with reports in the literature. Previous studies investigating the effects of MeHg on DA metabolism have primarily indicated that MeHg inhibits MAO activity (Beyrouty et al., 2006; Castoldi et al., 2006) . In these studies, MAO activity was measured in either whole or regional brain homogenates and therefore was not specific to DA neurons. As such, the observed decrease in MAO activity could have been due to collective changes in multiple types of cells, including glia. It is also plausible that the lack of change in MAO activity observed presently is specific to the undifferentiated PC12 cell line. For example, expression of different MAO and ALDH isozymes in nigrostriatal (NS) DA neurons, when compared with undifferentiated PC12 cells, could mediate alternative changes in the aberrant DA metabolomic profile induced by exposure to MeHg. Two isozymes of MAO, designated A and B, have been proposed based on substrate selectivity and inhibitor sensitivity (Cai et al., 1998) . MAO A has a higher affinity for DA, and is predominately found in rat catecholamine neurons and PC12 cells (Jahng et al., 1997; Naoi et al., 1987) . However, in most species both forms can oxidize DA (O’Carroll et al., 1983), and in humans MAO B is primarily responsible for DA oxidation (Riederer and Jellinger, 1982) . Similarly, 2 isozymes of ALDH, ALDH2, and ALDH1A1, can mediate DA metabolism (Marchitti et al., 2007) . In PC12 cells, ALDH2 catalyzes the oxidation of DOPAL, whereas in NSDA neurons both ALDH2 and ALDH1A1 can participate (Anderson et al., 2011; Galter et al., 2003) . Given these differences, an investigation of the aberrant DA metabolomic profile induced by MeHg in NSDA neurons is necessary. However, the observed changes in DA metabolites suggest that mechanisms by which MeHg targets DA metabolism are more complex than previously assumed. Evidence for Indirect Rather than Direct ALDH Inhibition in MeHg-Impaired DA Metabolism To discern direct versus indirect mechanisms by which MeHg may target ALDH, the DA metabolomic profile induced by MeHg was compared with that induced by daidzin, a reversible, selective direct ALDH2 inhibitor, and rotenone, a reversible inhibitor of Complex I of the mitochondrial ETC. While all 3 compounds reduced DOPAC concentrations to the same extent, the concomitant increase in DOPET was variable, with MeHg having a greater effect than daidzin or rotenone. Therefore, while all 3 compounds inhibit the “oxidative pathway” of DA metabolism, the mechanisms by which they do so differs. MeHg does not directly inhibit ALDH as daidzin does (Keung and Vallee, 1993) . This effect was confirmed by measuring ALDH enzymatic activity (Guru and Shetty, 1990). While daidzin was able to inhibit ALDH reversibly, MeHg had no effect on the enzyme’s activity. Therefore, an indirect mechanism of action by MeHg on ALDH is more likely to account for the changes in DA metabolism. Indirect inhibition of ALDH was evaluated by measuring mitochondrial respiration and cytosolic NADþ levels following treatment with either MeHg or rotenone. The rationale for this comparison was that rotenone directly inhibits Complex I of the mitochondrial ETC, which decreases availability of the ALDH cofactor NADþ and indirectly impairs ALDH activity (Lamensdorf et al., 2000a) . Presently, rotenone shifted DA metabolism from the oxidative to the reductive pathway suggesting that ALDH activity was indirectly inhibited. However, while rotenone almost completely abolished spare mitochondrial respiration, there was no change in basal respiration. Because rotenone is a reversible inhibitor of Complex I and was not present during measurements of mitochondrial respiration, it is likely that basal respiration was able to recover from insult. A more stringent measurement of mitochondrial bioenergetics, spare respiratory capacity, demonstrated that rotenone did induce stress and impair mitochondrial function overall. When compared with rotenone, MeHg inhibited both basal and spare mitochondrial respiration. This observation suggests that MeHg impairs mitochondrial respiratory capacity irreversibly. The level at which MeHg inhibits the mitochondrial ETC is likely downstream of Complex I at the level of either Complex II (Mori et al., 2011) or Complex III (Yee and Choi, 1996) . Inhibition of Complex III with antimycin decreases Complex I activity by up to 75% (Beattie et al., 1994), and as a result NADH conversion to NADþ would likely be compromised. In support of this hypothesis, NADþ levels were significantly depleted following exposure to MeHg. Thus, ALDH activity could still be inhibited secondarily by mitochondrial dysfunction at the level of Complex III following exposure to MeHg. However, the present data are only corollary. Additional experimentation is needed to demonstrate a causative relationship between MeHg-induced mitochondrial dysfunction, cofactor availability, and impaired ALDH enzymatic activity. Potential Biological Consequence of MeHg-Impaired DA Metabolism The observation that MeHg increases DOPAL accumulation is biologically relevant. Increased production of DOPAL is one mechanism by which MeHg may exert toxicity in DA synthesizing cells. DOPAL has demonstrated toxic effects in DA neurons both in vivo and in vitro. Microinjections of DOPAL in the substantia nigra cause focal, generalized lesions associated with a loss of TH immunoreactivity and other phenotypic neuronal markers, as well as marked gliosis (Burke et al., 2003) . In differentiated PC12 cells and striatal synaptosomes, exposure to micromolar concentrations of DOPAL increases the incidence of cell death (Mattammal et al., 1995) . In addition to increasing toxicity itself, DOPAL in synergy with glucose deprivation and mitochondrial ETC inhibition enhances toxicity (Lamensdorf et al., 2000a) . Hence, inhibition of DOPAL oxidation potentiates toxicity induced by metabolic stress and mitochondrial dysfunction. As demonstrated presently, MeHg impairs mitochondrial respiration and abolishes reserve respiratory capacity in PC12 cells. Therefore, the selective sensitivity of DA synthesizing cells to MeHg may result from a combination of the generalized molecular mechanism by which the toxicant exerts its effects, and the distinct cellular environment created by the presence of DA and the enzymes necessary for its metabolism. The finding that MeHg increases DOPAL activity as a result of indirect ALDH inhibition provides evidence in PC12 cells in support of the hypothesis that exposure to MeHg contributes to the etiology of Parkinson disease (PD). A link between ALDH inhibition and PD has previously been established following occupational exposure to another environmental toxicant. Casida et al. (2014) demonstrate that the fungicide benomyl causes concentration-dependent inhibition of ALDH, which is associated with increased production of DOPAL and DOPET, and decreased production of DOPAC in vivo in mouse striatum and in vitro in PC12 cells. In humans, occupational exposure to benomyl increases the incidence of PD by 67% (Fitzmaurice et al., 2013) . Therefore, environmental toxicants, including benomyl and MeHg, inhibit ALDH sufficiently to damage DA neurons and increase the risk of PD in exposed humans. Summary The present work demonstrates that MeHg alters the DA metabolic profile in undifferentiated PC12 cells and shunts DA metabolism along the alternative reductive metabolic pathway. Results suggest that ALDH activity is inhibited indirectly by mitochondrial dysfunction and decreased availability of the ALDH cofactor NADþ. Consequences of impaired DA metabolism contribute to accumulation of the toxic DA metabolic intermediate, DOPAL. 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Tiernan, Chelsea T., Edwin, Ethan A., Hawong, Hae-Young, Ríos-Cabanillas, Mónica, Goudreau, John L., Atchison, William D., Lookingland, Keith J.. Methylmercury Impairs Canonical Dopamine Metabolism in Rat Undifferentiated Pheochromocytoma (PC12) Cells by Indirect Inhibition of Aldehyde Dehydrogenase, Toxicological Sciences, 2015, 347-356, DOI: 10.1093/toxsci/kfv001