Kiningham, Ph.D., Research Assistant Professor, Graduate Center for
Toxicology, University of Kentucky, Lexington, KY. (E-mail: email@example.com).
Curt Pendergrass, Ph.D., President, ALT, Inc. and Assistant Director, TEST
Foundation, Lexington, KY (E-mail: firstname.lastname@example.org).
preliminary research is part of grant proposal recently submitted to the CAN Foundation
for funding, http://www.cureautismnow.org)
as a potential target of thimerosal toxicity.
binding proteins as potential targets of thimerosal toxicity.
of cellular antioxidant capacity for protection against thimerosal mediated
are potent neurotoxins. Studies
have demonstrated that mercury (Hg) can localize to both neuronal and glial
cell types in the central nervous system and elicit a range of deleterious
actions, many of which are attributable to the affinity of Hg for sulfhydryl
groups in proteins. Sodium
ethylmercuri-thiosalicylate (Thimerosal) is a widely used Hg containing
antiseptic and preservative in topical medications, cleaning solutions for eye
lenses, cosmetics and vaccines. Metabolism of thimerosal generates ethylmercury, a more
lipophilic and therefore potentially more reactive mercurial than either
methyl or inorganic Hg. Although
specific cellular targets of thimerosal induced toxicity have not been
identified, it is conceivable that the mitochondria, an organelle whose
function depends on sulfhydryl containing enzymes might be a target of this
Studies by Bucio et
al. (1) and Brawer et al. (2) suggest that mitochondria accumulate mercuric
chloride and methyl mercury, leading to an alteration in organelle
architecture and function. Mitochondria are the most abundant cellular
organelles and are the predominant site of cellular energy generation (ATP).
Therefore, mitochondrial dysfunction can have significant ramifications
within a cell. Mitochondrial
damage can be amplified into cell death by triggering apoptotic pathways.
Organic mercurials such as methyl mercury have been shown to cause
apoptosis in various cell types including rat cerebellar neurons and T cells (3,4). The mechanism of organic
mercurial-dependent apoptosis has not been elucidated; however, all forms of
Hg have been shown to modulate intracellular calcium (Ca+2)i and
down regulate the activities of cellular antioxidants such as glutathione,
glutathione peroxidase or superoxide dismutases. Mitochondria are of central importance for maintenance of
cellular redox status and physiological Ca+2 homeostasis.
An increase in (Ca+2)i can lead to an increase in
reactive oxygen species (ROS) and reactive nitrogen species (RNS) production both in
the cytosol and mitochondria. The
ROS/RNS can directly interact and modify cellular components such as proteins
and DNA leading to toxicity and cell death.
Within the mitochondria an increase in ROS/RNS production without
sufficient antioxidant defenses could lead to inhibition of respiratory chain
enzymes culminating in energy depletion and cell death.
An increase in ROS production can alter the redox status of a cell and
failure to balance the oxidative stress can trigger commitment to apoptotic
cell death through modulation of gene expression.
hypothesize that the mitochondria is a target for thimerosal induced toxicity.
This hypothesis will be tested with three specific aims:
Aim 1 will determine if the mechanism of thimerosal mediated toxicity is
through the mitochondria and whether or not it represents a more potent
mitochondrial toxin than methyl mercury.
Metabolism of thimerosal into the more lipophilic ethylmercury would enhance
partitioning into the cell and possibly the mitochondria compared to methyl
An established neuroblastoma cell line, SK-N-SH, will be treated with
increasing concentrations of thimerosal or methyl mercury and the
incorporation of mitochondrial Hg measured by cold vapor atomic absorption.
In addition the mode (apoptosis vs. necrosis) and mechanism of cell
death will be determined. Apoptotic
vs. necrotic cell death will be distinguishable using the following
approaches: electron microscopy, DNA fragmentation analysis, flow cytometric
analysis of apoptotic specific markers as well as caspase enzyme activity.
In preparation for
this study we have performed preliminary
experiments to evaluate the toxicity of thimerasol.
Thimerasol treatment (100 nM - 10 µM)
resulted in dose dependent cell death within 24 hours of exposure.
Furthermore, experiments were performed to gain mechanistic insight
into thimerasol toxicity. A
dose dependent increase in the DNA binding activity of the redox sensitive
transcription factor NFkB was observed. AP-1 DNA binding activity, which is a redox sensitive
transcription factor implicated in neuronal cell death, was increased with 10 µM
treatment. These results suggest that ROS production as a result of thimerosal
treatment may contribute to cell death in our model.
To our knowledge, this type of analysis has not been performed in
evaluation of mercurial-induced cytotoxicity and provides clues of potential
types of genes that may be modulated to play a role in thimerosal-induced cell
death. Identification of genes
which may be altered as a result of thimerosal exposure may provide potential
pharmacological targets to which Hg toxicity could be attenuated.
Figure 1 is a gel mobility shift assay from a neuroblastoma cell
line (SK-N-SH) treated with increasing concentrations of thimerosal (0-10 µM)
for 30 min to 4 hours. Nuclear extracts were prepared and 10 µg of protein was incubated with a
radiolabeled oligonucleotide corresponding to a consensus element of NFkB.
A time and
dose dependent increase in NFkB
DNA binding activity was observed in the
thimerasol treated cells. An increase in NFkB occurs in response to
cellular stress, generally thought to occur as a result of an increase in
reactive oxygen species production. At the 4 hour time
point the increase in NFkB was observed only in the 10
µM treated cells. This concentration resulted in apoptotic cell death within
24 hours. Therefore it is possible that at low concentrations, an
increase in NFkB would result in up regulation of protective proteins; however, beyond a critical
threshold (5-10 µM), NFkB
signals apoptotic cell death.
Figure 2 is photograph of a DNA ladder as a result
of thimerosal treatment.: A neuroblastoma cell line (SK-N-SH) was treated
without (lane 2) or with 10 µM thimerosal (lane 3) for 24 hours. DNA was
isolated and separated on a 1.5% agarose gel. The banding pattern in lane
3 which consists of numerous DNA fragments is characteristic of apoptosis.
Lane 1 is a 1 kB DNA ladder loading control.
Specific Aim 2
Specific Aim 3 will determine if
enhancement of cellular antioxidant capacity would attenuate thimerosal
1) Published studies show that over expression of the mitochondrial
antioxidant, manganese superoxide dismutase (MnSOD), can protect against cell
death as a result of exposure to nitric oxide generating agents, alkalosis, or
inhibition of respiration (9,10,11); 2) We
have preliminary data to show that over expression of MnSOD can prevent
apoptotic cell death as a result of (Ca+2)i
imbalance; 3) Methyl mercury toxicity has been shown to be attenuated by
over expression of MnSOD (12); 4) Methyl
mercury induced increases in ROS formation in the brain is inhibited by the
metal chelator deferoxamine (13); 5) Administration of melatonin to the SHSY5Y
neuroblastoma cell line protected cells against mercuric chloride induced
glutathione depletion and mitochondrial injury (14). Taken together, these
studies suggest that modulation of the intracellular antioxidant capacity can
attenuate Hg induced toxicity.
Our laboratory has the expertise of using genetic approaches to enhance
overall cellular and/or mitochondrial antioxidant capacity.
The use of these techniques in combination with application of
pharmacological agents which either exhibit mitochondrial selectivity (MnSOD
mimetic, creatine, ubiquinone) or represent a more general antioxidant (melatonin,
N-acetylcysteine, deferoxamine) will be used to ascertain the most
efficient way to attenuate thimerosal toxicity.
A few studies have suggested that an enhanced overall antioxidant
capacity can protect against inorganic or methylmercury toxicity.
This study will determine if targeting of antioxidants specifically to
the mitochondria would be more effective in reducing mercurial toxicity,
particularly with more lipophilic compounds such as thimerosal.
:Significant progress has been made in furthering our understanding of
thimerosal induced toxicity by the aforementioned experiments; however,
additional studies are needed to identify specific cellular targets of
thimerosal induced damage. For example, changes in cellular transcription
factors can result in changes in protein expression. Apoptosis, as noted in the
neuroblastoma cell line upon thimerasol treatment may be correlated with the
increase in NFkB which was
noted in specific aim #1. Apoptosis is a mode of cell death which may be stopped
if the initial signal is identified. Under some circumstances apoptosis is
beneficial; however, because neurons cannot divide, an apoptotic cell is lost
forever and therefore the function of the central nervous system is compromised.
Additional studies in our laboratory which are not depicted here
show that neurons are especially sensitive to thimerosal-induced toxicity when
compared to other cell types. Identification of neuronal specific targets is
therefore vital in understanding the susceptibility of this cell type to
thimerosal mediated damage. The technique of nucleotide photoaffinity labeling
provides a unique and sensitive approach to identification of specific cellular
molecules which are altered as a result of thimerosal treatment. We believe our
approach to studying thimerosal toxicity represents a unique collaborative
effort which, upon proper funding, could further elucidate specific cellular
targets to which the toxicity of thimerosal could be attenuated.
This proposal represents a unique collaboration between scientists whose
expertise are in the fields of oxidative stress and Hg toxicity. Based on our
preliminary experiments, we believe that our approach will further our
knowledge or thimerosal induced toxicity and will identify ways in which this
can be attenuated.
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