The Effects of Methylmercury on Mitochondrial Function and
Reactive Oxygen Species Formation in Rat Striatal Synaptosomes
Anne Dreiem,*,1Caitlyn C. Gertz,* and Richard F. Seegal*,†
*New York State Department of Health, Wadsworth Center, Albany, New York, 12201, and †School of Public Health,
University at Albany, Albany, New York, 12222
Methylmercury (MeHg) is especially toxic to the developing
central nervous system. In order to understand the reasons for this
age-dependent vulnerability, we compared the effects of MeHg on
formation of reactive oxygen species (ROS) and mitochondrial
function in striatal synaptosomes obtained from rats of various
ages. Basal ROS levels were greater, and basal mitochondrial
function was lower, in synaptosomes from younger animals,
compared to adult animals. MeHg induced ROS formation in
synaptosomes from rats of all ages, although the increases were
greatest in synaptosomes from the younger animals. MeHg also
reduced mitochondrial metabolic function, as assessed by MTT
reduction, as well as mitochondrial membrane potential; again,
the greatest changes were seen in synaptosomes from early
postnatal animals. These age-dependent differences in suscepti-
bility to MeHg are most likely due to a less efficient ROS
detoxifying system and lower activity of mitochondrial enzymes
in tissue from young animals.
Key Words: methylmercury; synaptosomes; reactive oxygen
species; mitochondria; development.
Methylmercury (MeHg), an environmental pollutant, is
known to be highly neurotoxic, especially to the developing
nervous system (National Research Council, 2000). At present,
the main exposure route for MeHg is via food, especially
through the consumption of fish and fish products. Exposure to
high concentrations of MeHg is known to have serious effects
on brain development, as seen after the catastrophic poisoning
episodes in Japan and Iraq (National Research Council, 2000).
The effects of chronic, low-dose exposure are more controver-
sial, but in two large epidemiological studies, one from the
Faroe Islands (Grandjean et al., 1997) and one from New
Zealand (Kjellstro ¨m et al., 1986, 1989), prenatal exposure to
low MeHg levels from maternal consumption of fish was
associated with neurodevelopmental deficits in children. How-
ever, in another study from the Seychelles Islands, there was no
such association (Davidson et al., 1998; Myers et al., 2003).
At present, the reasons for the greater sensitivity of the
developing nervous system to MeHg are not well understood.
Differences in toxicokinetics and toxicodynamics lead to
greater accumulation of MeHg in fetal brain than in maternal
brain. Even beyond these differences, however, the developing
nervous system also appears to be more sensitive to MeHg, due
to factors intrinsic to the brain (Choi, 1989). Elucidation of the
mechanisms involved in MeHg toxicity would aid in un-
derstanding the greater sensitivity of the developing nervous
system. Although such studies have been performed both in
adult animals and in invitro test systems, mechanisms of MeHg
toxicity have not, to the best of our knowledge, been compared
in developing and adult animals.
A number of mechanisms and molecular targets have been
proposed to be involved in MeHg neurotoxicity, including
alterations in calcium homeostasis (Komulainen and Bondy,
1987; Marty and Atchison, 1997), binding to sulfhydryl groups
(Hughes, 1957), and apoptosis/necrosis (Kunimoto, 1994).
However, in the recent years, several studies have implicated
the formation of reactive oxygen species, or ROS, (Ali et al.,
1992; LeBel et al., 1990; Sarafian and Verity, 1991; Yee and
Choi, 1994, 1996) and disruption of mitochondrial function
(Bondy and McKee, 1991; Hare and Atchison, 1992; Limke
and Atchison, 2002) as two key mechanisms in MeHg-induced
neuronal damage. We hypothesized that differences in ROS
production and/or defense mechanisms, and mitochondrial
enzyme activities contribute to the greater sensitivity of
developing animals to MeHg, compared to adult animals. To
test this hypothesis, we have compared ROS formation and
mitochondrial function after MeHg exposure in synaptosomes
from postnatal day 7, 14, or 21 rats to that of adult rats.
MATERIALS AND METHODS
Materials. Adult male and timed-pregnant Long Evans rats were obtained
from Taconic Farms (Germantown, NY). Methylmercuric chloride (98%) was
purchased from EM Sciences (Gibbstown, NJ). Methylthiazoletetrazolium
(MTT) and hydrogen peroxide (H2O2) were purchased from Sigma-Aldrich
(St. Louis,MO).2#,7#-Dichlorofluorescin-diacetate (DCFH-DA) and 5,5#,6,6#-
tetrachloro-1,1#,3,3#-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) were
purchased from Molecular Probes (Eugene, OR). Pierce BCA protein assay
1To whom correspondence should be addressed at New York State
Department of Health, Wadsworth Center, Empire State Plaza, Albany, NY
12201. Fax: (518) 486-1505. E-mail: email@example.com.
? The Author 2005. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
For Permissions, please email: firstname.lastname@example.org
TOXICOLOGICAL SCIENCES 87(1), 156–162 (2005)
Advance Access publication June 15, 2005
by guest on June 3, 2013
reagents were purchased from Pierce Biotechnology, Inc. (Rockford, IL). All
other chemicals were analytical grade.
Preparation of striatal synaptosomes. All procedures involving animals
were performed according to the protocols of the Wadsworth Center In-
stitutional Animal Care and Use Committee. Striatal synaptosomes were
prepared essentially as described previously (Andersen et al., 2003). Briefly,
naı ¨ve male LongEvans rats were sacrificed at an ageof 10–14weeks (adults)or
on postnatal day (PND) 7, 14, or 21. For PND 7–21 rats, animals were collected
from different litters. The brains were removed and placed on ice, and the
striata were quickly dissected. The striata from the animals of the same age
were pooled and homogenized on ice in 10 volumes of 0.32 M sucrose in
a Potter-Elvehjem homogenizer. The homogenate was centrifuged at 4?C for
10 min at 600 3 g in a Beckman TJ-6 centrifuge. The supernatant was then
diluted 1:1 with 1.3 M sucrose, to obtain a suspension at a final concentration of
0.8 M sucrose. This suspension was further centrifuged at 20,000 3 g for
30min at4?C, resultingin a myelin-richsupernatantand a pellet(P2) consisting
of synaptosomes and free (extrasynaptosomal) mitochondria. The supernatant
was discarded, and the pellet was resuspended in oxygenated HEPES buffer
(pH 7.4). The synaptosomes were kept on ice until the experiments were
performed, usually within 15–20 min.
AssessmentofROS formation. DCFH-DA,anonfluorescentcell-permeable
compound, diffuses passively across cell membranes. Once inside the cell,
the acetate groups are cleaved by intracellular esterases, yielding 2#,7#-
dichlorofluorescin (DCFH). DCFH can be oxidized by hydroxyl radicals,
peroxynitrite, or H2O2 (in the presence of peroxidases) to a fluorescent
compound, 2#,7#-dichlorofluorescein (DCF) (Myhre et al., 2003).
Synaptosomes were diluted 1:40 in HEPES buffer before loading with
10 lM DCFH-DA for 15 min at 37?C. DCFH-DA stock solutions (10 mM in
dimethylformamide) were made fresh daily. After incubation, the synapto-
somes were pelleted by centrifugation (1200 3 g for 7 min), and the buffer was
replaced with fresh HEPES buffer only.
Working solutions of MeHg or H2O2(the latter used as a positive control)
were prepared daily by diluting stock solutions in HEPES buffer to 1.673 the
desired final concentration, and 150 ll of the working solutions were placed in
each well of a 96-well microplate (Costar). The reaction was started by the
addition of 100 ll of the synaptosome solution to each well (final reaction
volume 250 ll). Four wells were used for each condition. Each plate was
incubated in a shaker incubator at 37?C for 30 min before fluorescence was
measured using a Perkin Elmer LS50 spectrometer (excitation wavelength 488
nm, emission wavelength 525 nm, band widths 5 nm). Blank values were
obtained from wells containing buffer and synaptosomes that had not been
loaded with DCFH-DA. Synaptosomal protein levels were determined with
Pierce BCA reagents according to provided instructions. DCF fluorescence
values were corrected for protein levels and autofluorescence of the samples,
according to the formula
where Fcois corrected fluorescence value, Fsais observed fluorescence in the
sample, and Fblis observed fluorescence in the blank.
Assessment of mitochondrial metabolic function. Mitochondrial meta-
bolic function was assessed by the conversion of the dye methylthiazolete-
trazolium (MTT) to formazan. This assay is based on the ability of the
mitochondrial enzyme succinate dehydrogenase to metabolize MTT into
formazan, a reaction that takes place only in functionally intact mitochondria.
Working solutions of MeHg were prepared as described above. P2synapto-
somes were prepared as described for the ROS assay, but because protein
contents were lower in the synaptosome fractions from the younger rats, the
synaptosomes were diluted in HEPES buffer to 1:20 for PND 7 rats, 1:27 for
PND 14 rats, and 1:40 for PND 21 and adult rats; these dilutions provided
similar protein levels for samples from animals of differing ages. 150 ll of the
working solutions were aliquoted into microtubes, and 100 ll of the
synaptosome suspension and 25 ll of a 5.0 mg/ml solution of MTT in HEPES
buffer wereaddedto eachtube.Thesampleswereincubatedfor30 minat37?C.
The purple formazan crystals were pelleted by centrifugation, and the
supernatant discarded. The pellets were dissolved in DMSO and transferred
to 96-well microplates (Falcon). The formation of formazan was quantitated
spectrophotometrically at 570 nm using an EL808 Ultra Microplate Reader
(Bio-Tek Instruments, Inc). Data were expressed as percentage of age-matched
Mitochondrial membrane potential. Mitochondrial membrane potential
(Dwm) was measured using the fluorescent dye JC-1 (5,5#,6,6#-tetrachloro-
1,1#,3,3#- tetraethylbenzimidazolylcarbocyanine iodide). This dye exhibits
a fluorescence shift from red to green, due to disassembly of dye aggregates,
when the mitochondrial membrane potential decreases (Reers et al., 1991).
Synaptosomes were prepared as described above and diluted 1:40 in HEPES
buffer. The synaptosomes were then incubated with MeHg in HEPES buffer at
37?C for 30 min. The synaptosomes were pelleted by centrifugation (1500 3 g,
4?C) for 5 min, the buffer was replaced with fresh buffer without MeHg, and
JC-1 (stock solution 0.5 mM in DMSO) was added to a final concentration of
0.5 lM. After 20 min incubation at 37?C, fluorescence was measured in an
EPICS’EliteESP flowcytometer (CoulterCorp.),usinga 525 ±10nmstandard
filter for detection of green fluorescence and a custom-made 595 ± 10 nm filter
(Omega Optical, Inc) for detection of orange fluorescence. For each sample,
10,000 events (synaptosomes) were analyzed. Due to day-to-day variations in
fluorescence intensity, data were expressed as percentage of age-matched
Statistical analysis. Data were compared for significant differences by
analysis of variance (ANOVA). When the overall test of significance lead to
rejection of the null hypothesis, a post hoc test was performed to determine
the source of the effects. Basal levels for ROS and MTT were compared
by Bonferroni’s test, whereas the effects of MeHg on ROS, MTT, and Dwm
were analyzed by Dunnett’s test. Differences were considered significant
when p ? 0.05.
ROS levels in nonexposed synaptosomes were dependent on
the age of the donor rats (Fig. 1). The highest background
levels were found in synaptosomes from young animals (5.2 ±
0.8, 6.3 ± 0.6, and 4.8 ± 0.5 fluorescence units per lg protein
for PND 7, 14, and 21 animals, respectively), and the lowest
levels in synaptosomes from adult animals (2.6 ± 0.1). ROS
levels were significantly higher in synaptosomes from the
young rats (PND 7, 14, and 21) than in synaptosomes from the
adult; however, there were no significant differences in ROS
levels among the younger animals.
MeHg-exposure increased ROS formation in synaptosomes
from both adult and young animals (Fig. 2), with the highest
ROS levels observed in synaptosomes from younger animals.
The sensitivity to MeHg-induced increases in ROS was, in
decreasing order: PND 7 > PND 14 > PND 21 > adult rat
synaptosomes. In synaptosomes from PND 7 rats, MeHg
concentrations as low as 1.0 lM gave a small, but significant,
increase in ROS formation. For synaptosomes from PND 14 or
older rats, 2.5 lM MeHg was needed to induce statistically
significant increases in ROS. Univariate analysis of variance
showed that there were significant effects of both age and
MeHg on ROS levels (correlation coefficients 0.721 and 0.935,
AGE-DEPENDENT EFFECTS OF METHYLMERCURY
by guest on June 3, 2013
respectively; p ? 0.001), as well as a significant interaction
between age and MeHg concentration (correlation coefficient
0.620; p ? 0.001).
It is possible that different properties of synaptosomes from
young and adult rats could influence the uptake or deester-
ification of DCFH-DA, and that this could account for the
differences in ROS formation that we observed among
synaptosomes from rats of differing ages. To investigate this
possibility, we used H2O2as a positive control. Because H2O2
is cell permeable (Halliwell and Gutteridge, 1999), it crosses
the synaptosomal membrane and oxidizes deesterified DCFH
independently of intracellular ROS-generating mechanisms.
We found that 100 lM H2O2increased ROS formation to the
same extent (2–2.6 fluorescence units/lg protein) in prepara-
tions from rats of all ages (Table 1), demonstrating that
differences in the effects of MeHg on ROS formation among
the preparations were not due to differences in intrasynapto-
somal DCFH concentration.
Mitochondrial Metabolic Function
Basal levels for the reduction of MTT to formazan were
dependent on the age of the donor rats (Fig. 3). The mean ± SE
was 2.5 ± 0.3 for PND 7, 3.2 ± 0.2 for PND 14, 4.8 ± 0.2 for
PND 21, and 4.1 ± 0.2 for adult rat synaptosomes (expressed as
A570corrected for protein contents of the samples). Because of
these differences, results were expressed as percentage of age-
matched vehicle (DMF) controls.
MeHg exposure reduced mitochondrial metabolic function
in synaptosomes from rats of all ages (Fig. 4), with significant
reductions observed after exposure to MeHg at 2.5 lM or
greater concentrations in synaptosomes from PND 7, PND 14,
and PND 21 rats. Concentrations of MeHg ? 5 lM were
needed to reduce mitochondrial function in adult rat synapto-
somes. At 10 lM MeHg, MTT metabolism in all synaptosomal
preparations was reduced to 25–35% of age-matched controls.
Univariate analysis of variance showed that MTT reduction
was highly dependent on MeHg concentration (correlation
coefficient 0.917, p ? 0.001). There was also a significant
younger rats than in tissue from adults. Striatal synaptosomes were loaded with
DCFH-DA and incubated for 30 min without MeHg before background ROS
formationwas assessedby measuring increasein DCF fluorescence. The results
are given in fluorescence units, corrected for protein content in the synapto-
somes. Each value is the mean of 16 to 22 wells in three to four independent
experiments ± SE. *Significantly different from adult (p < 0.05, ANOVA,
ROS levels in striatal synaptosomes are higher in tissue from
Effects of Hydrogen Peroxide on ROS Formation in
Synaptosomes from Long Evans Rats of Various Ages
100 lM H2O2
5.2 ± 0.8
6.3 ± 0.6
4.8 ± 0.4
2.6 ± 0.1
7.2 ± 0.7
8.9 ± 0.7
7.2 ± 0.5
5.1 ± 0.2
Note. Each value given is the mean of 16–22 wells in 3–4 independent
experiments ± SE.
from younger rats. Striatal synaptosomes were loaded with DCFH-DA and
exposed to MeHg for 30 min before ROS formation was assessed by measuring
increase in DCF fluorescence. The results are given as percentage of age-
matched vehicle controls (controls are set to 100%). Each value is the mean of
16 to 22 wells in three to four independent experiments ± SE. *Significantly
different from age-matched controls (p < 0.05, ANOVA, Dunnett’s test).
MeHg induces greater ROS formation in striatal synaptosomes
than in tissue from adults. Mitochondrial function was assessed by reduction of
MTT to formazan in untreated synaptosomes from rats of different ages.
Formation of formazan was determined by measuring absorbance at 570 nm,
and the values were adjusted for the protein amount of the samples. Each value
is the mean of eight or nine wells in three independent experiments ± SE.
*Significantly different from PND 7; #significantly different from PND 14
(p < 0.05, ANOVA, Bonferroni’s test).
Basal activity in MTT assay is lower in tissue from younger rats
DREIEM, GERTZ, AND SEEGAL
by guest on June 3, 2013
effect of age and an interaction between age and MeHg
concentration, with correlation coefficients of 0.336 and
0.396, respectively (p ? 0.001 and p ¼ 0.004).
Mitochondrial Membrane Potential
MeHg reduced Dwmin synaptosomes from rats of all ages
(Fig. 5); 0.1 lM MeHg reduced Dwmto 75 ± 8 and 73 ± 6% of
control levels in synaptosomes from PND 7 and PND 21 rats,
respectively. Small reductions were also observed at this
exposure level in synaptosomes from PND 14 and adult rats,
but these did not reach statistical significance. After exposure
to 0.5 lM MeHg, large decreases in Dwmwere observed in all
preparations; however, the effects were much more pronounced
in synaptosomes from PND 7 and PND 14 animals, for which
Dwms were reduced to 17 ± 4 and 29 ± 7% of control levels,
respectively. In synaptosomes from PND 21 and adult animals,
0.5 lM MeHg reduced Dwmto 57 ± 10 and 60 ± 7% of control
levels. With higher MeHg exposures (1.0–2.5 lM) the Dwm
was completely abolished in synaptosomes from rats of all
ages. Univariate analysis of variance showed a strong correla-
tion between MeHg concentration and reduction of Dwm
(correlation coefficient 0.981; p ? 0.001). There was also
a significant correlation between age and reduction of Dwm
and an interaction between age and MeHg concentration
(correlation coefficients 0.448 and 0.716, respectively; p ¼
0.007 and p ? 0.001).
In the present study we have shown that basal ROS levels are
higher in synaptosomes from young animals than in those from
adults. It is well known that the fetus and the neonate have
lower levels of antioxidants and enzymes that protect against
ROS than do adult animals (Buonocore et al., 2001). Mavelli
and coworkers found that Mn-superoxide dismutase (MnSOD)
activity was almost absent in the brains of 7-day-old rats, while
the activity increased to about 90% of adult levels at 3 weeks of
age (Mavelli et al., 1982). Similarly, catalase and cytosolic
Cu,ZnSOD levels also increased with age, although, in general,
catalase levels in brain are very low, and the changes in
Cu,ZnSOD levels were less prominent than with MnSOD
(Mavelli et al., 1982). Less efficient scavenging of ROS by
antioxidants and enzymes such as SOD could therefore be
a reason for the higher basal ROS levels found in synaptosomes
from younger animals.
The finding that basal ROSlevelsare higher in synaptosomes
development and increased in the adult rat. This discrepancy
could be due to the fact that Driver and coworkers used a crude
homogenate preparation that contained glial fractions, as glia
contains ROS scavengers, such as metallothionein and gluta-
thione, and are important for maintenance of GSH levels in
neurons (Dringen et al., 2000; Kohler et al., 2003).
Our results also show that MeHg exposure increases ROS
formation in synaptosomes in vitro. These results are in
agreement with previous findings that MeHg exposure in-
creases oxidative stress, both in vivo in rodent brain (Ali et al.,
1992; Yee and Choi, 1994) and in several in vitro systems,
including primary cultures of rat cerebellar granule cells,
cerebral cortical neurons, astrocytes, and oligodendrocytes,
and in synaptosomes from whole brain (except cerebellum)
(Bondy and McKee, 1990; Shanker and Aschner, 2003; Yee
and Choi, 1996). Although the exposure times in our experi-
ments were considerably shorter than those used in cell-culture
function in synaptosomes from younger rats. Striatal synaptosomes were
exposed to MeHg for 30 min, and mitochondrial function was then assessed by
conversion of MTT to formazan. The results are given as percentage of age-
matched vehicle controls (controls are set to 100%). Each value is the mean of
eight or nine wells in three independent experiments ± SE. *Significantly
different from control (p < 0.05, ANOVA, Dunnett’s test).
MeHg induces greater decrease in mitochondrial metabolic
younger rats. Synaptosomes were exposed to MeHg before Dwmwas assessed
by JC-1 fluorescence. The results are given as percentage of age-matched
vehicle controls(controlsare set to 100%).Eachvalue is the mean± SEofthree
to four independent experiments. *Significantly different from age-matched
controls (p < 0.05, ANOVA, Dunnett’s test).
MeHg decreases Dwmto a greater extent in synaptosomes from
AGE-DEPENDENT EFFECTS OF METHYLMERCURY
by guest on June 3, 2013
experiments, the MeHg concentrations required to induce ROS
formation are in the same range (1–2.5 lM), demonstrating that
striatal synaptosomes are highly sensitive to the toxic effects
Most importantly, the increases in ROS seen after MeHg
exposure were dependent on the age of the donor rats, with the
highest ROS levels seen in synaptosomes from the youngest
animals. To the best of our knowledge, age-related differences
in ROS formation after MeHg exposure in rat tissue have not
been reported previously. There are several possible explan-
ations for our observations. First, as already mentioned, young
animals have lower levels of antioxidants and less active ROS
detoxifying enzymes than do adults, which may result in
a reduced capability to clear cells of toxic ROS once they are
formed. Furthermore, in a recent study it was shown that
prenatal exposure to MeHg decreased antioxidant enzyme
activity (Vicente et al., 2004). Second, it is possible that ROS
generating systems are more active in tissue from young
animals, resulting in higher ROS levels. Although the route
of MeHg-induced ROS formation is presently unknown,
mitochondria are believed to be key targets for MeHg (Hare
and Atchison, 1992) and are important sites of ROS formation
(Halliwell and Gutteridge, 1999). An increased capacity for
ROS formation in the mitochondria of the young animals could
render the system more vulnerable to the effects of MeHg.
Furthermore, these mechanisms are not exclusive, so a combi-
nation of increased ROS formation and less ROS detoxification
could synergistically cause the large increases in ROS levels in
Mitochondrial Metabolic Function
Basal levels of MTT reduction were significantly lower in
synaptosomes from the youngest rats, compared to prepara-
tions from older rats. This age-dependent difference is most
likely due to lower activity of succinate dehydrogenase (SDH),
the enzyme primarily responsible for reduction of MTT, in the
mitochondria of young rats (Pollak and Duck-Chong, 1973;
Potter et al., 1945). The activity of SDH in brain is constant
from about 3 days before birth until about 6 days after birth and
then increases rapidly to the adult level within 30 days (Potter
et al., 1945).
These differences in basal activity may aid in explaining the
observation that MeHg decreased mitochondrial metabolic
function in synaptosomes, with the greatest effects seen in
preparations from the youngest animals. If there is a minimum
SDH activity needed to maintain normal mitochondrial func-
tion, then a lower basal SDH activity in the young animals may
cause reduced tolerance to toxicants that further decrease SDH
activity. The low basal SDH activity in tissue from young rats
may therefore contribute to the higher sensitivity of the
developing brain to MeHg.
Our finding that MeHg reduces mitochondrial metabolic
function in the MTT assay is in agreement with results of
a previous study in primary cerebellar granule cells, where
exposure to 2.5 lM MeHg for 1 h reduced MTT metabolism to
approximately 45% of control levels (Castoldi et al., 2000). In
the adult brain, the cerebellar granule cell is the cell type with
the highest sensitivity to MeHg; these cells die at concen-
trations at which surrounding cells like astrocytes and Purkinje
cells are spared (Hunter and Russel, 1954). However, after
developmental MeHg exposure, damage is more widely
distributed, and severe degeneration and atrophy can be present
throughout the cerebellum and the cerebral hemispheres
(Eccles and Annau, 1987). Furthermore, previous experiments
in our laboratory have shown reduced levels of dopamine in
striatal synaptosomes after in vitro exposure to MeHg (Shan
and Seegal, unpublished data), and there is evidence that
methylmercury affects the activity of the plasma membrane
dopamine transporter in rat striatal tissue (Faro et al., 2002).
Therefore, we chose to investigate effects of MeHg on
synaptosomes from the striatum. Our results indicate that
striatal synaptic terminals also are highly sensitive to
Mitochondrial Membrane Potential (Dwm)
The results of the present study demonstrate that MeHg
exposure leads to a reduction in Dwmin mitochondria, with the
greatest effects seen in tissue from the younger animals. It is
known that the activity of several mitochondrial enzymes is
low at birth and increases with age in rats; these enzymes
include (in addition to SDH) glutamate dehydrogenase, malate
dehydrogenase, isocitrate dehydrogenase, cytochrome oxidase,
and ATP phosphohydrolase (Pollak and Duck-Chong, 1973;
Potter et al., 1945). A lower basal activity of such enzymes in
the neonate, relative to adults, could mean that the added insult
of MeHg will have a greater effect on mitochondria from
younger animals. For example, cytochrome oxidase (complex
IV) is an enzyme of the mitochondrial electron transport chain
that contributes to the electrochemical gradient across the inner
mitochondrial membrane. This enzyme is inhibited by methyl-
and ethylmercury (Bickar et al., 1984; Mann and Auer, 1980;
Usuki et al., 1998), probably due to interactions of mercury
with essential sulfhydryl groups. It is possible that the low
basal activity of this enzyme in young animals renders
mitochondria more vulnerable to MeHg-induced inhibition of
cytochrome oxidase, contributing to age-dependent differences
Reductions in Dwmmay influence neuronal survival, as cells
induced to undergo apoptosis show an early reduction in Dwm
prior to exhibiting other apoptotic markers, such as DNA
fragmentation or externalization of phosphatidylserine. Com-
plete disruption of Dwmis only found in cells that are destined
to die by apoptosis or necrosis (Kroemer et al., 1997). The
complete loss of Dwmin our experiments, therefore, indicates
that MeHg exposure, even at very low concentrations, could be
sufficient to induce neuronal death in young animals. Although
DREIEM, GERTZ, AND SEEGAL
by guest on June 3, 2013
apoptosis is a cellular phenomenon and thus cannot be studied
entirely in synaptosomes, this observation is in agreement with
findings of previous studies, in which 0.1–3.0 lM MeHg was
shown to induce apoptosis in various cell types in culture,
including cerebellar granule cells from rats and mice, neuro-
blastoma cells, and PC12 cells (Bulleit and Cui, 1998; Castoldi
et al., 2000; Kunimoto, 1994; Miura et al., 1999).
Our results also demonstrate that Dwmis a very sensitive
endpoint for MeHg toxicity, since significant reductions were
observed even after exposure to MeHg concentrations as low as
0.1 lM. These results are in agreement with an earlier study by
Limke and Atchison (2002), who found that mitochondria in
primary cultures of rat cerebellar granule cells depolarize
irreversibly after 50-min exposure to 0.5 lM MeHg. In
contrast, Castoldi and coworkers did not observe effects of
1 lM MeHg on Dwmfor up to 4–6 h (Castoldi et al., 2000).
Differences between these studies may be due to methodology,
since in the study by Castoldi and coworkers, the loss of
membrane potential was not assessed quantitatively. Neverthe-
less, our results clearly indicate that the striatal synaptosome
preparation is highly sensitive to MeHg-induced effects.
In addition to the mechanisms investigated in the present
study, several other mechanisms have been suggested for
MeHg-induced brain damage. These include inhibition of
glutamate uptake in astrocytes, with subsequent rise in
extracellular glutamate and excitotoxicity, and alterations in
intracellular calcium levels (for review see Costa et al., 2003).
In vivo, such mechanisms may operate in concert to cause the
symptoms of MeHg poisoning. For example, increased cyto-
solic calcium can lead to calcium accumulation in the
mitochondria and depolarization of the inner mitochondrial
membrane, leading to loss of the proton gradient and opening
of the mitochondrial permeability pore. Increased intracellular
calcium can also lead to formation of ROS by activation of
enzymes such as nitric oxide synthase, leading to formation of
nitric oxide (Halliwell and Gutteridge, 1999). Further research
is necessary to elucidate the role of these mechanisms in
MeHg-induced developmental neurotoxicity.
In conclusion, we have shown that ROS are produced, and
that mitochondrial function and Dwmare reduced, in striatal
synaptosomes from rats after exposure to MeHg. The use of
synaptosomes as a model system allows the comparison of
preparations made from rats at different developmental stages,
and although the effects are present in tissue from both young
and adult rats, they are most prominent in preparations from the
youngest animals. This demonstrates that factors intrinsic to
the brain, and not toxicokinetics alone, contribute to the high
sensitivity of the developing brain to MeHg. From the present
study it cannot be determined whether MeHg-induced in-
creases in ROS levels are a cause or a consequence of
mitochondrial damage; further experiments are currently being
undertaken to elucidate this. In any case, the lower basal
activities in ROS-detoxifying mechanisms and mitochondrial
enzymes, either alone or in combination, are likely to
contribute to the observed age-related differences in sensitivity
The authors thank Dr. David A. Lawrence and Renjie Song for helpful
discussions and excellent technical assistance. This work was supported by
NIEHS/USEPA Centers for Children’s Environmental Health and Disease
Prevention Research Grants 1P01ES11263 and EPA grant R829390. Conflict
of interest: none declared.
Ali, S. F., LeBel, C. P., and Bondy, S. C. (1992). Reactive oxygen species
formation as a biomarker of methylmercury and trimethyltin neurotoxicity.
Neurotoxicology 13, 637–648.
Andersen, J. M., Myhre, O., and Fonnum, F. (2003). Discussion of the role of
the extracellular signal-regulated kinase-phospholipase A2 pathway in
production of reactive oxygen species in Alzheimer’s disease. Neurochem.
Res. 28, 319–326.
Bickar, D., Bonaventura, J., Bonaventura, C., Auer, H., and Wilson, M. (1984).
Paradoxical effects of methylmercury on the kinetics of cytochrome c
oxidase. Biochemistry 23, 680–684.
Bondy, S. C., and McKee, M. (1990). Prevention of chemically induced
synaptosomal changes. J. Neurosci. Res. 25, 229–235.
Bondy, S. C., and McKee, M. (1991). Disruption of the potential across the
synaptosomal plasma membrane and mitochondria by neurotoxic agents.
Toxicol. Lett. 58, 13–21.
Bulleit, R. F., and Cui, H. (1998). Methylmercury antagonizes the survival-
promoting activity of insulin-like growth factor on developing cerebellar
granule neurons. Toxicol. Appl. Pharmacol. 153, 161–168.
Buonocore, G., Perrone, S., and Bracci, R. (2001). Free radicals and brain
damage in the newborn. Biol. Neonate 79, 180–186.
Castoldi, A. F., Barni, S., Turin, I., Gandini, C., and Manzo, L. (2000). Early
acute necrosis, delayed apoptosis and cytoskeletal breakdown in cultured
cerebellar granule neurons exposed to methylmercury. J. Neurosci. Res. 59,
Choi, B. H. (1989). The effects of methylmercury on the developing brain.
Prog. Neurobiol. 32, 447–470.
Costa, L. G., Aschner, M., Vitalone, A., Syversen, T., Soldin, O. P. (2003).
Developmental neuropathology of environmental agents. Annu. Rev. Phar-
macol. Toxicol. 44, 87–110.
Davidson, P. W., Myers, G. J., Cox, C., Axtell, C., Shamlaye, C., Sloane-
Reeves, J., Cernichiari, E., Needham, L., Choi, A., Wang, Y., et al. (1998).
Effects of prenatal and postnatal methylmercury exposure from fish
consumption on neurodevelopment: Outcomes at 66 months of age in the
Seychelles Child Development Study. JAMA 280, 701–707.
Dringen, R., Gutterer, J. M., and Hirrlinger, J. (2000). Glutathione metabolism
in the brain. Eur. J. Biochem. 267, 4912–4916.
Driver, A. S., Kodavanti, P. R. S., and Mundy, W. R. (2000). Age-related
changes in reactive oxygen species production in rat brain homogenates.
Neurotoxicol. Teratol. 22, 175–181.
Eccles, C. U., and Annau, Z. (1987). Prenatal exposure to methylmercury. In
The Toxicity of Methyl Mercury (C. U. Eccles and Z. Annau. Eds.), pp. 114–
130. Johns Hopkins University Press, Baltimore, MD.
Faro, L. R., do Nascimento, J. L., Alfonso, M., and Duran, R. (2002).
Mechanism of action of methylmercury on in vivo striatal dopamine
release. Possible involvement of dopamine transporter. Neurochem. Int. 40,
AGE-DEPENDENT EFFECTS OF METHYLMERCURY
by guest on June 3, 2013
Grandjean, P., Weihe, P., White, R. F., Debes, F., Araki, S., Yokoyama, K.,
Murata, K., Sorensen, N., Dahl, R., and Jorgensen, P. J. (1997). Cognitive
deficit in 7-year-old children with prenatal exposure to methylmercury.
Neurotoxicol. Teratol. 19, 417–428.
Halliwell, B., and Gutteridge, J. M. C. (1999). Free Radicals in Biology and
Medicine, 3rd ed., pp. 1–936. Oxford University Press, New York.
Hare, M. F., and Atchison, W. D. (1992). Comparative action of methylmer-
cury and divalent inorganic mercury on nerve terminal and intraterminal
mitochondrial membrane potentials. J. Pharmacol. Exp. Ther. 261,
Hughes, W. L. (1957). A physicochemical rationale for the biological activity
of mercury and its compounds. Ann. N.Y. Acad. Sci. 65, 454–460.
Hunter, D., and Russel, D. S. (1954). Focal cerebellar and cerebellar atrophy
in a human subject due to organic mercury compounds. J. Neurochem. 17,
Kjellstro ¨m, T., Kennedy, P., Wallis, S., and Mantell, C. (1986). Physical and
mental development of children with prenatal exposure to mercury from fish.
Stage I: Preliminary tests at age 4. National Swedish Environmental
Protection Board Report 3080. Solna, Sweden.
Kjellstro ¨m, T., Kennedy, P., Wallis, S., Stewart, A., Friberg, L., Lind, B.,
Wutherspoon, T., and Mantell, C. (1989). Physical and mental development
of children with prenatal exposure to mercury from fish. Stage II: Interviews
and psychological tests at age 6. National Swedish Environmental Protection
Board Report 3642. Solna, Sweden.
Kohler, L. B., Berezin, V., Bock, E., and Penkowa, M. (2003). The role of
metallothionein II in neuronal differentiation and survival. Brain Res. 992,
Komulainen, H., and Bondy, S. C. (1987). Increased free intrasynaptosomal
Ca2þby neurotoxic organometals: Distinctive mechanisms. Toxicol. Appl.
Pharmacol. 88, 77–86.
Kroemer, G., Zamzami, N., and Susin, S. A. (1997). Mitochondrial control of
apoptosis. Immunol. Today 18, 44–51.
Kunimoto, M. (1994). Methylmercury induces apoptosis of rat cerebellar
neurons in primary culture. Biochem. Biophys. Res. Commun. 204, 310–317.
LeBel, C. P., Ali, S. F., McKee, M., and Bondy, S. C. (1990). Organometal-
induced increases in oxygen reactive species: The potential of 2#,7#-
dichlorofluorescin diacetate as an index of neurotoxic damage. Toxicol.
Appl. Pharmacol. 104, 17–24.
Limke, T. L., and Atchison, W. D. (2002). Acute exposure to methylmercury
opens the mitochondrial permeability transition pore in rat cerebellar granule
cells. Toxicol. Appl. Pharmacol. 178, 52–61.
Mann, A. J., and Auer, H. E. (1980). Partial inactivation of cytochrome c
oxidase by nonpolar mercurial reagents. J. Biol. Chem. 255, 454–458.
Marty,M. S.,andAtchison,W.D.(1997).PathwaysmediatingCa2þentry in rat
cerebellar granule cells following in vitro exposure to methyl mercury.
Toxicol. Appl. Pharmacol. 147, 319–330.
Mavelli, I., Rigo, A., Federico, R., Ciriolo, M. R., and Rotilio, G. (1982).
Superoxide dismutase, glutathione peroxidase and catalase in developing rat
brain. Biochem. J. 204, 535–540.
Miura, K., Koide, N., Himeno, S., Nakagawa, I., and Imura, N. (1999). The
involvement of microtubular disruption in methylmercury-induced apoptosis
in neuronal and nonneuronal cell lines. Toxicol. Appl. Pharmacol. 160,
Myers, G. J., Davidson, P. W., Cox, C., Shamlaye, C. F., Palumbo, D.,
Cernichiari, E., Sloane-Reeves, J., Wilding, G. E., Kost, J., Huang, L. S.,
et al. (2003). Prenatal methylmercury exposure from ocean fish consumption
in the Seychelles child development study. Lancet 361, 1686–1692.
probes 2#,7#-dichlorofluorescin diacetate, luminol, and lucigenin as indicators
of reactive species formation. Biochem. Pharmacol. 65, 1575–1582.
National Research Council (2000). Toxicological Effects of Methylmercury,
National Academy Press, Washington, DC.
Pollak, J. K., and Duck-Chong, C. G. (1973). Changes in rat liver mitochondria
and endoplasmic reticulum during development and differentiation. Enzyme
Potter, V. R., Schneider, B. S., and Liebl, G. J. (1945). Enzyme changes during
growth and differentiation in the tissues of the newborn rat. Cancer Res. 5,
Reers, M., Smith, T. W., and Chen, L. B. (1991). J-aggregate formation of
a carbocyanine as a quantitative fluorescent indicator of membrane potential.
Biochemistry 30, 4480–4486.
Sarafian, T., and Verity, M. A. (1991). Oxidative mechanisms underlying
methyl mercury neurotoxicity. Int. J. Dev. Neurosci. 9, 147–153.
Shanker, G., and Aschner, M. (2003). Methylmercury-induced reactive oxygen
species formation in neonatal cerebral astrocytic cultures is attenuated by
antioxidants. Brain Res. Mol. Brain Res. 110, 85–91.
Usuki, F., Yasutake, A., Matsumoto, M., Umehara, F., and Higuchi, I. (1998).
The effect of methylmercury on skeletal muscle in the rat: A histopatholog-
ical study. Toxicol. Lett. 94, 227–232.
Vicente, E., Boer, M., Netto, C., Fochesatto, C., Dalmaz, C., Siqueira, I. R.,
Goncalves, C.-A. (2004). Hippocampal antioxidant system in neonates from
methylmercury-intoxicated rats. Neurotoxicol. Teratol. 26, 817–823.
Yee, S., and Choi, B. H. (1994). Methylmercury poisoning induces oxidative
stress in the mouse brain. Exp. Mol. Pathol. 60, 188–196.
Yee, S., and Choi, B. H. (1996). Oxidative stress in neurotoxic effects of
methylmercury poisoning. Neurotoxicology 17, 17–26.
DREIEM, GERTZ, AND SEEGAL
by guest on June 3, 2013