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Thimerosal Neurotoxicity

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Thimerosal Neurotoxicity

Video Tapes are now available of Jane M. El-Dahr, M.D.,  Head of Pediatric Allergy/
Rheumatology,  Tulane University Health Sciences Center lecturing on "Autism and immunology effects---The heavy metal connection."


Boyd E. Haley, Ph.D., Professor and Chairman, Chemistry Department, University of Kentucky, lecturing on "The biochemical interrelationships of Vitamin C, melatonin, glutathione and other redox compounds."

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).


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


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


Specific Aim 2 will identify specific enzymes in the cell with emphasis on mitochondrial localization which are targets of thimerosal toxicity.  


Rationale:  We have previously reported that Hg is a potent inhibitor of a number of important nucleotide binding proteins in the human brain (5,6).  Treatment of human control brain homogenates with low (1-3 µM) concentrations of Hg in vitro induces aberrant protein-nucleotide interactions characteristic of Alzheimer’s Disease (AD), a disease where brain Hg levels have been reported to be elevated (7).  Moreover, we have shown that 2 week exposure of rats to elemental Hg vapor at levels present in the mouths of persons with high numbers of amalgam fillings results in the same aberrant protein-nucleotide interactions found in AD (8).  Recently our laboratory has found that thimerosal is capable of inhibiting the activity of a number of critical mammalian nucleotide binding proteins (including but not limited to:  creatine kinase, adenylate kinase, phosphoglycerate kinase, pyruvate kinase) in vitro at low (< 5 µM) levels, a fraction of what is actually included in many vaccines (see Thimerosal Toxicity Slide Show).. 


Approach:  The technique of nucleotide photoaffinity labeling with P-32 labeled analogs has been used in the past for the study of toxicant induced changes in protein nucleotide interactions.  The use of this technique in conjunction with one and two-dimensional electrophoresis, autoradiography, radioanalytic detection and Western blot analysis will allow for the identification of specific targets of thimerosal toxicity. 


Significance:  The significance of this aim will be in the identification of specific enzymes to which a rapid assay could be developed as a marker of thimerosal toxicity.  


Figure 3A.  SDS-PAGE analysis of neuroblastoma cell homogenates photolabeled with [32P]2N3ATP and [32P]8N3ATP after in vitro treatment with increasing concentrations of thimerosal for 24 hrs.



Figure 3B.  Autoradiogram made from SDS-PAGE showing decreased [32P]2N3ATP and [32P]8N3ATP photolabeling of multiple ATP binding proteins after in vitro treatment with increasing concentrations of thimerosal for 24 hrs.



Figures 4A-4D. Plot of the quantified data from the experiments shown in Figure 3.


4A)                                                          4C)


4B)                                                        4D)


The results of experiments shown in Figure 3 and plotted in Figure 4, show that photolabeling of many ATP binding proteins found in neuroblastoma cells was decreased significantly following in vitro treatment with increasing concentrations of thimerosal for only 24 hrs.  For example, [g32P]2N3ATP photolabeling of a 60 kDa protein band (red arrow) and a 26 kDa protein band (orange arrow) were decreased by 53% and 50% respectively, after 5 µM thimerosal treatment relative to the control.  At the same time, [g32P]8N3ATP photolabeling of a 90 kDa (blue arrow) and 48 kDa (green arrow) protein bands were decreased  by 57% and 28% respectively relative to the control.


Figure 5A & 5B.  SDS-PAGE (A) and autoradiogram (B) made from neuroblastoma cell homogenates photolabeled with [32P]8N3GTP after in vitro treatment with increasing concentrations of thimerosal for 24 hrs.



Figures 6A-6D. Plot of the quantified data from the experiment shown in Figure 5.


6A)                                                          6C)


6B)                                                           6D)


The results of experiments shown in Figure 5 and plotted in Figure 6, show that photolabeling of many GTP binding proteins found in neuroblastoma cells is altered significantly following in vitro treatment with increasing concentrations of thimerosal for only 24 hrs.  For example, [g32P]8N3GTP photolabeling of a 92 kDa protein band (green arrow) and a 46 kDa protein band (orange arrow) were decreased by 26% and 43% respectively, after 5 µM thimerosal treatment relative to the control.  In contrast, [g32P]8N3GTP photolabeling of a 50 kDa (blue arrow) and 18 kDa (red arrow) protein bands were increased  by 64% and 72% at 5 µM thimerosal respectively, relative to the control.


Summary and Conclusions



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 of 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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