Methylmercury Impairs Canonical Dopamine Metabolism in Rat Undifferentiated Pheochromocytoma (PC12) Cells by Indirect Inhibition of Aldehyde Dehydrogenase
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.,
. 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
(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
(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)
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
(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
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
thus could bind directly to cysteine residues present in the
active site of either MAO
(Hubalek et al., 2003)
(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
(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)
effects of MeHg have been previously described in this
(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
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
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
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
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.
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
(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
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)
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
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
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)
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.
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
(Bemis and Seegal, 1999; Dreiem et al., 2009)
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
(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
(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.,
. 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,
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
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
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
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
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)
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)
differentiated PC12 cells and striatal synaptosomes, exposure to
micromolar concentrations of DOPAL increases the incidence of
(Mattammal et al., 1995)
. In addition to increasing
toxicity itself, DOPAL in synergy with glucose deprivation and
mitochondrial ETC inhibition enhances toxicity
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%
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.
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. While additional experimentation is necessary
to validate the present observations in DA neurons in vivo, these
data provide evidence for a mechanism by which DA neurons
may be selectively sensitive to the toxic effects of MeHg.
Supplementary data are available online at http://toxsci.
The authors gratefully acknowledge the advice and
technical assistance of Drs Ravindra Hajela and Sara Ciotti.
National Institutes of Health (ViCTER supplement to
R01ES03299 and R25NS006577).
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