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: firstname.lastname@example.org.
? The Author 2005. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
For Permissions, please email: email@example.com
TOXICOLOGICAL SCIENCES 87(1), 156–162 (2005)
Advance Access publication June 15, 2005
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