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In Vitro Treatment of a Neuroblastoma Cell Line with Low Concentrations of Thimerosal Results in Apoptosis and Altered Nucleotide Binding


Kelley Kiningham, Ph.D., Research Assistant Professor, Graduate Center for Toxicology, University of Kentucky, Lexington, KY. (E-mail: kkkini0@pop.uky.edu).


J. Curt Pendergrass, Ph.D., President, ALT, Inc. and Assistant Director, TEST Foundation, Lexington, KY (E-mail: cpender@altcorp.com).


(This preliminary research is part of grant proposal recently submitted to the CAN Foundation for funding, http://www.cureautismnow.org)




Specific Aim 1

Mitochondria as a potential target of thimerosal toxicity.

Specific Aim 2

Nucleotide binding proteins as potential targets of thimerosal toxicity.

Specific Aim 3

Enhancement of cellular antioxidant capacity for protection against thimerosal mediated toxicity.

Summary and Conclusion


References Cited


Mercurials 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 toxin.  


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.


We hypothesize that the mitochondria is a target for thimerosal induced toxicity.  This hypothesis will be tested with three specific aims: 


Specific 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.


Rationale: Metabolism of thimerosal into the more lipophilic ethylmercury would enhance partitioning into the cell and possibly the mitochondria compared to methyl mercury. 


Approach:   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

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.

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 toxicity.  


Rationale:  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.  


Approach:  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.  


Significance:  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.


Summary. :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.


Conclusion.  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.

References Cited


 1.  Bucio, L., Garcia, C., Souza, V., Hernandez, E., Gonzalez, C., Betancourt, M., and Gutierrez-Ruiz, M.C. (1999) Uptake, cellular distribution and DNA damage produced by mercuric chloride in a human fetal hepatic cell line.  Mutat. Res. 423(1-2), 65-72.


 2.  Brawer, J.R., McCarthy, G.F., Gornitsky, M., Frankel, D., Mehindate, K., and Schipper, H.M. (1998) Mercuric chloride induces a stress response in cultured astrocytes characterized by mitochondrial uptake of iron.  Neurotoxicology 19(6), 767-776.


 3.  Kunimoto, M. (1994) Methylmercury induces apoptosis of rat cerebellar neurons in primary culture.  Biochem. Biophys. Res. Commun. 204(1), 301-317.


 4.  Shenker, B.J., Guo, T.L., and Shapiro, I.M. (1998)  Low-level methylmercury exposure causes human T-cells to undergo apoptosis:  evidence of mitochondrial dysfunction.  Environ. Res.  77, 149-159.


 5.  Pendergrass, J.C., and Haley, B.E. (1996)  Inhibition of brain tubulin-guanosine 5’-triphosphate interactions by mercury:  Similarity to observations in Alzheimer’s disease.  In:  Metal Ions in Biological Systems (Sigel, H. and Sigel, A., eds.)  pp 461-478.  Marcel Dekker, Inc. New York.


 6.  Pendergrass, J.C. and Haley, B.E. (1995)  Mercury-EDTA complex specifically blocks brain b- tubulin-GTP interactions:  similarity to observations in Alzheimer’s Disease.  In Status Quo and Perspectives of Amalgam and Other Dental Materials (Friberg, L.T. and Schrauzer, G.N., eds.) pp. 98-105.  Georg Thieme Verlag, Stuttgart.


 7.  Duhr, E.F., Pendergrass, J.C., Slevin, J.T., and Haley, B.E. (1993).  HgEDTA complex inhibits GTP interactions with the E-site of brain b-tubulin.  Toxicology and Applied Pharmacology 122, 273-280.


 8.  Pendergrass, J.C., Haley, B.E., Vimy, M.J., Winfield, S.A., and Lorscheider, F.L. (1997)  Mercury vapor inhalation inhibits binding of GTP to tubulin in rat brain:  Similarity to a molecular lesion in Alzheimer diseased brain.  Neurotoxicology 18, 315-324.


 9.  Gonzalez-Zuleta, M., Ensz, L.M., Mukhina, G., Lebovitz, R.M., Azacka, R.M., Engelhardt, J.F., Oberley, L.W., Dawson, V.L., and Dawson, T.M. (1998) Manganese superoxide dismutase protects nNOS neurons from NMDA and nitric oxide-mediated neurotoxicity.  J. Neurosci. 18, 2040-2055.


10. Majima, H.J., Oberley, T.D., Furukawa, K., Mattson, M.P., Yen, H.C., Sweda, L.I. and St. Clair, D.K. (1998) Prevention of mitochondrial injury by manganese superoxide dismutase revels a primary mechanism for alkaline-induced cell death.  J. Biol. Chem. 273, 8217-8224.


11. Kiningham, K.K., Oberley, T.D., Lin, S.M., Mattingly, C.A., and St. Clair, D.K. (1999) Overexpression of manganese superoxide dismutase protects against mitochondrial initiated poly(ADP-ribose) polymerase-mediated cell death. FASEB J. 13, 1601-1610.  


12. Naganuma, A., Miura, K., Tanaka-Kagawa, T., Kitahara, J., Seko, Y., Toyoda, H., and Imura, N. (1998) Overexpression of manganese-superoxide dismutase prevents methylmercury toxicity in Hela cells.  Life Sci. 62(12), 157-161.


13. LeBel, C.P., Ali, S.F., and Bondy, S.C. (1992) Deferoxamine inhibits methyl mercury-induced increases in reactive oxygen species formation in rat brain.  Toxicology and Applied Pharmacology.  112, 161-165.


14. Olivieri, G., Brack, C., Muller-Spahn, F., Stahelin, H.B., Herrmann, M., Renard, P., Brockhaus,  M., and Hock, C. (2000) Mercury induces cell cytotoxicity and oxidative stress and increases b-amyloid secretion and tau phosphorylation in SHSY5Y neurobloastoma cells. J. Neurochem. 74, 231-236.  

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