They will, and they should, because it is.
The Age of Autism: Mercury ban opposed
By DAN OLMSTED
Representatives of 22 medical organizations have written to all members of Congress opposing efforts to ban the mercury-based preservative thimerosal from vaccines.
"Our organizations respectfully wish to state our opposition to all legislative efforts at the federal and state levels to restrict access to vaccines containing thimerosal, an ethylmercury-based preservative," said the letter dated April 3 from "Multiple National Organizations that Support Safe and Effective Vaccines."
The groups said that banning the preservative in vaccines for children and pregnant women -- as several states have done and legislation in Congress proposes -- would "perpetuate false and misleading information that vaccines are not safe. Parents may see the banning of thimerosal as an admission that vaccine safety oversight is inadequate."
In fact, the letter said, "There has been considerable research on this issue since the 1999 precautionary statement of the U.S. Public Health Service and the American Academy of Pediatrics and there is no documented scientific evidence that ethylmercury in the form of thimerosal in the doses administered in vaccines causes any risk to health."
The letter also cited concerns that bans could trigger "ongoing vaccine shortages or inability to deliver care. ... Limit the nation's inability to quickly administer influenza vaccine in the U.S. when a pandemic strikes. ... Lead to increased costs for vaccines. ... Add more complexity to our present vaccine delivery system. ... Profoundly affect global immunization programs, as do many U.S. vaccine policy decision."
At issue are concerns raised by parents and some scientists that increasing exposure to thimerosal in childhood vaccines during the 1990s may have triggered a huge rise in autism diagnoses. In 1999 the Centers for Disease Control and Prevention and others recommended manufacturers phase out thimerosal as soon as possible to limit exposure.
In 2004 the Institute of Medicine of the National Academies said it found no connection and that future research should go to "more promising" areas.
Yet concerns have persisted, in part because some flu vaccines still contain thimerosal, and the CDC has recommended the vaccines for all pregnant women and for children ages 6 months to 5 years.
Those concerns have prompted several states -- including New York, Illinois, California, Iowa, Delaware and most recently Washington state -- to enact bans over the opposition of the CDC and state medical associations.
At the same time, pressure has mounted for more studies of potential health problems of thimerosal and vaccines in general. Last week U.S. Rep. Carolyn Maloney, D-N.Y., said she will introduce a bill this month to force the federal government to study the autism rate in never-vaccinated American children.
In a letter to Congressional health policy staff that accompanied the groups' statement opposing a thimerosal ban, Diane C. Peterson of the Immunization Action Coalition said: "As you may be aware, recent media attention has been given to the role of thimerosal in vaccines and the development of autism. The 22 national organizations that have signed this letter, as well as many others, stand behind the enormous amount of scientific evidence that shows no link exists between thimerosal in vaccines and the development of autism.
"Please oppose all anti-thimerosal legislative proposals and help further (the) nation's work in protecting children and adults against vaccine-preventable diseases."
The signers include representatives of the following groups:
Ambulatory Pediatric Association; American Academy of Family Physicians; American Academy of Physician Assistants; American College of Allergy, Asthma, and Immunology; American College of Preventive Medicine; American Liver Foundation; American Medical Directors Association; American Pharmacists Association; Association of Immunization Program Managers; Council of State and Territorial Epidemiologists; Every Child by Two; Hepatitis B Foundation; Hepatitis Foundation International; Immunization Action Coalition; Infectious Diseases Society of America; National Coalition on Adult Immunization; National Foundation for Infectious Diseases; Parents of Kids with Infectious Diseases; Pediatric Infectious Disease Society; Society for Adolescent Medicine; Society of Teachers of Family Medicine; Vaccine Education Center at the Children's Hospital of Philadelphia.
Here is the Letter:
Ambulatory Pediatric Association American Academy of Family Physicians
American Academy of Physician Assistants
American College of Allergy, Asthma, and Immunology
American College of Preventive Medicine American Liver Foundation
American Medical Directors Association American Pharmacists Association
Association of Immunization Program Managers
Council of State and Territorial Epidemiologists Every Child by Two
Hepatitis B Foundation Hepatitis Foundation International
Immunization Action Coalition Infectious Diseases Society of America
National Coalition on Adult Immunization National Foundation for Infectious Diseases
Parents of Kids with Infectious Diseases Pediatric Infectious Diseases Society
Society for Adolescent Medicine Society of Teachers of Family Medicine
Vaccine Education Center at the Children’s Hospital of Philadelphia
Date: April 3, 2006
To: All Members of Congress
From: Multiple National Organizations that Support Safe and Effective Vaccines
Subject: Opposition to Efforts to Restrict Access to Vaccines
Our organizations respectfully wish to state our opposition to all legislative efforts at the federal and state levels to restrict access to vaccines containing thimerosal, an ethylmercury-based preservative. If enacted, we believe such legislation has the potential to do the following:
1. Perpetuate false and misleading information that vaccines are not safe. Parents may see the banning of thimerosal as an admission that vaccine safety oversight is inadequate. The issue of thimerosal’s ill effects on the neurologic development of infants is based on studies of methylmercury and not the ethylmercury that is in the preservative thimerosal used in some vaccines. According to the U.S. Environmental Protection Agency, nearly all methylmercury exposures in the U.S. occur through eating fish and shellfish. The mercury that is contained in the preservative thimerosal is known as ethylmercury. There has been considerable research on this issue since the 1999 precautionary statement of the U.S. Public Health Service (USPHS) and the American Academy of Pediatrics (AAP) and there is no documented scientific evidence that ethylmercury in the form of thimerosal in the doses administered in vaccines causes any risk to health.
2. Potentially result in on-going vaccine shortages or inability to deliver care as healthcare providers are forced to seek vaccine formulations that are either free of thimerosal or contain only reduced quantities both of which would be in short supply. As an example, only 10% of a projected total of 80 million doses of injectable influenza vaccine will be available for the 2005-06 vaccination season in a thimerosal-free formulation. Other vaccines, such as vaccine used to prevent Japanese encephalitis in travelers to certain Asian countries, are not available in reduced thimerosal or thimerosal-free formulations.
3. Limit the nation’s ability to quickly administer influenza vaccine in the U.S. when a pandemic strikes. Vaccine containing no thimerosal or reduced quantities can be packaged only in single-dose units, and we are far short of the capacity necessary to fill enough single-dose units to quickly respond to a nation in need of immediate protection against influenza at the pandemic level (e.g., Avian flu). The only way we can more quickly build our vaccine delivery capacity is to fill multidose vials and these vials must contain a thimerosal-containing preservative.
4. Lead to increased costs for vaccines. Where alternative vaccines containing no thimerosal or only reduced quantities are available, they can be as much as 25-30% higher in cost, due to production losses and to single dose packaging. These additional costs will directly impact Medicare, the federal Vaccines for Children Program, state-administered Medicaid programs, as well as private health insurance costs.
5. Add more complexity to our present vaccine delivery system. With new vaccines being introduced, changes in vaccination scheduling, and all of the other complexities of vaccination delivery, it is already difficult for providers to stay current with the ever-changing nature of immunization. Adding a requirement that providers can only use vaccines with no or reduced amounts of thimerosal would add more complexity.
6. Profoundly affect global immunization programs, as do many U.S. vaccine policy decisions. Vaccines sold in the international market require multi-dose packaging because it reduces manufacturing costs significantly, a vital consideration for nations with fewer resources than the U.S. Multidose vials also conserve space in refrigerated containers (vaccines often require refrigeration when shipped to remote areas). If the U.S. adopts a policy restricting access to vaccines, it could adversely affect the health and well-being of children all over the world in ways that you would not intend. The negative political consequences of the U.S. using vaccines “allegedly safer” than those it supports for other countries are very worrisome. Vaccine manufacturers have revised their manufacturing processes to allow production of most vaccines in either a reduced thimerosal or thimerosal-free formulation. This was done as a precaution to address theoretical concerns noted in the USPHS/AAP joint request of July, 1999 and not because any evidence suggested that thimerosal was harmful. One fact we know for certain: in the U.S., 10.5 million cases of vaccine-preventable disease and 33,000 deaths are prevented each year by vaccinations. We therefore urge the members of the U.S. House of Representatives and the U.S. Senate to trust in the conclusions of the scientific community, including the Institute of Medicine, that the scientific evidence does not identify any connection between vaccines and autism. Please oppose all such legislative proposals and help us further our work in protecting our nation’s children and adults against vaccine-preventable diseases.
Diane Kittridge, MD, President
Ambulatory Pediatric Association
Mary E. Frank, MD, Board Chair
American Academy of Family Physicians
Marie-Michelle Leger, MPH, PA-C
Clinical and International Affairs
American Academy of Physician Assistants
William K. Dolen, MD, President
American College of Allergy, Asthma, and
David A. Smith, MD, President
American Medical Directors Association
Mitchel C. Rothholz, RPh, Vice President
Professional Practice & Member Services
American Pharmacists Association
Paul Bonta, Associate Director
Policy & Government Affairs
American College of Preventive Medicine
Marie P. Bresnahan, MPH
Vice President for Programs
American Liver Foundation
Dan Hopfensperger, Chair
Association of Immunization Program Managers
C. Mack Sewell, MD, President
Council of State and Territorial Epidemiologists
Amy Pisani, Executive Director
Every Child by Two
Molli C. Conti, Vice President of Community Outreach
Hepatitis B Foundation
Thelma King Thiel, Chair and CEO
Hepatitis Foundation International
Deborah L. Wexler, MD,
Executive Director Immunization Action Coalition
Martin J. Blaser, MD, President
Infectious Diseases Society of America
Kristin L. Nichol, MD, Chair
National Coalition for Adult Immunization
Len Novick, Executive Director
National Foundation for Infectious Diseases
Trish Parnell, Executive Director
Parents of Kids with Infectious Diseases
Joseph W. St. Geme, III, MD, President
Pediatric Infectious Diseases Society
Robert T. Brown, MD, President
Society for Adolescent Medicine
Donald Middleton, MD, Chair
Group on Immunization Education and
William Mygdal, EdD, President
Society of Teachers of Family Medicine
Paul A. Offit, MD, Center Director
Vaccine Education Center
Children’s Hospital of Philadelphia
The National Autism Association and SafeMinds have responded to the pro-mercury letter:
Opposition to Anti-Thimerosal Legislation
We have just received a copy of a letter that has been sent to the majority of health care staffers on Capitol Hill that promotes the continued use of thimerosal in vaccines. Incredibly, this letter has been endorsed by a large number of health care organizations. We must be ready to discuss this during our meetings in Washington this week. We find it highly unlikely to be coincidental that this letter was sent just before our rally and meetings with legislators to discuss the thimerosal/autism connection.
We've prepared arguments against continued use of thimerosal that can be printed out, along with the pro-mercury letter from Offit et al, and taken with you to any meetings you've arranged with your legislators. We hope this will help you be prepared to discuss the health threats from use of mercury-containing vaccines with your representative's staff.
Date: April 4th, 2006
To: All Members of Congress
From: Multiple Scientists and National Autism Organizations
Subject: Support for efforts to remove neurotoxin mercury from vaccines administered to pregnant women and children
Honorable Members of the 109th Congress
We feel legislation specifically delineating the removal of thimerosal (ethyl mercury) from vaccines is necessary in an effort to restore public confidence in vaccines and to prevent the potential for injury in our most vulnerable citizens; the fetus, infants and children. We base our concerns with the policy of exposing pregnant women and children to ethyl mercury on scientific facts and current policy.
The purpose of this letter is to make known our support for the efforts to remove ethyl mercury, a known neurotoxin, from vaccine preparations currently recommended for administration to pregnant women, infants and children.
1. The EPA recently reported that 1 in every 6 women of childbearing age is predicted to already have levels of mercury in their bodies that could cause neurodevelopmental harm to their unborn children. Of additional concern is the fact that the unborn fetus is known to accumulate mercury at levels higher than their mother by as much as 70%. Pregnant women are therefore counseled to avoid mercury exposure from seafood (methyl mercury) due to these concerns. Toxicological data recently provided by NIH funded research found that ethyl mercury, the type in the vaccine preservative thimerosal may be even more dangerous to the developing brain than methyl mercury. In this investigation it was documented that ethyl mercury resulted in twice as much inorganic mercury deposited in the brains of infant primates who received equal amounts of both ethyl mercury and methyl mercury modeled to mimic the early infant vaccine schedule from the 1990’s. Inorganic mercury in the brains of primates is known to result in neuroinflammation, a finding recently documented in brain tissue from those suffering with autism.
2. All flu vaccine manufacturers have a mercury-free or reduced mercury product. Sanofi, one of the leading flu vaccine producers is on record stating that they can make enough mercury-free vaccine to meet the needs of infants and pregnant women. Sanofi has also said that the amount of mercury-free product they produce is based on demand, so if more purchasers request it, they will increase production.
3. In December 2005 a provision was added to the Defense Bill giving Health & Human Services Secretary Michael Leavitt the ability to override state bans on mercury in the event of a human pandemic. Therefore, state and Federal legislation banning the use of thimerosal would be suspended should a pandemic occur. In addition, FDA does require the use of a preservative in multi-dose vials. There are several FDA approved preservatives, including 2- phenoxyethanol currently used in the Infanrix DTaP vaccine, that could be utilized that do not contain mercury.
4. According to vaccine manufacturers, the cost for a thimerosal free flu vaccine is approximately $3.30 or the cost of a “Happy Meal” from McDonalds. Although costs are a concern, American children deserve to receive the safest and most effective vaccine available, which clearly would be one that does not contain mercury. For example, the amount of mercury in a vial of flu vaccines that contains thimerosal is equal to a concentration of 50,000 parts per billion (ppb). To put this in perspective, liquid waste that exceeds 200 ppb of mercury must be disposed of in a special hazardous waste landfill and drinking water cannot exceed 2 ppb mercury. Unused flu vaccine must be disposed of as a hazardous waste. One must ask if most Americans would want to their infant to be the recipient of a product that can be classified as a hazardous waste?
5. Although the argument has been made that recommending thimerosal free vaccines for pregnant women, infants and children would increase the complexity of the vaccine schedule, complexity is something that health care providers deal with successfully on a daily basis. At present health care providers are dealing with a barrage of phone calls and questions from consumers who are desperate to find mercury free flu vaccines for their children. Should all flu vaccine be mercury free this issue would become a moot point for consumers and heath care providers alike.
6. Vaccine recommendations and formulations vary by country and in some countries the Government is the vaccine manufacturer. In fact, pharmaceutical companies have continued to sell off their mercury-based supplies to third world countries. When thimerosal was removed from vaccines in the United States, vaccine manufacturers even made a generous donation to the Global Alliance for Vaccines and Immunization of their thimerosal-containing vaccines. Global Policy in the U.S does not dictate policy in other countries.
In summary: The National Academy of Sciences acknowledges “windows of vulnerability” to mercury toxicity during neurological development. Specific types of neurodevelopmental outcomes may have different (and specific) windows. These critical periods for mercury effects have not been established and may be relatively short in duration. Because thimerosal from vaccines has been documented to cross the blood brain barrier and result in significant accumulation of inorganic mercury in the brains of infant primates, excessive exposure during one or more windows of neurodevelopmental vulnerability may have occurred. The fact that thimerosal may contribute to adverse neurodevelopmental outcomes is compounded by the recognition that even relatively minor effects early in life can have profound affects on society when amortized across the entire population and life span.
Therefore; in the interest of precaution, removal of mercury from vaccines given to vulnerable populations is warranted and actions that lead to such removal, especially since sufficient supplies of mercury free vaccines are readily available, should be actively supported.
Attached is a brief summary for the Committee of the recently published research on thimerosal at low doses close to or equal to that found in vaccines or at concentrations that are likely to result from vaccine administration.
Note: the mercury dose from vaccines produces acute ethylmercury blood levels in the nanomolar range. The half life is 5-7 days, meaning that half the injected dose of mercury leaves the blood in that time period, on average. There is considerable individual variation. Any background mercury exposures from non-vaccine sources would increase the blood mercury levels.
1. Baskin (2003) – thimerosal disrupts cell membranes, damages DNA, and alters cell shape at concentrations only 4 times those expected from vaccines. Greater effects were seen as the length of time of exposure grew, suggesting that under real conditions the concentration needed for the observed alterations would be much lower. This has been shown in subsequent research, that exposure of cells to nanomolar levels of thimerosal after 24 hours results in cell alterations.
2. Burbacher (2005): infant monkeys dosed with vaccine-level thimerosal were compared with infant monkeys dosed with equal levels of methylmercury. The thimerosal dose resulted in lower blood levels but more than twice the inorganic, or long term, mercury levels in the brain, relative to the methylmercury. The study showed the potential for significant brain accumulation from thimerosal and demonstrated that exposure/safety assessments for methylmercury may not apply to thimerosal.
3. Havarinasab & Hultman (2005): thimerosal given to mice alters immune function more than equal doses of methylmercury.
4. Hornig (2005): dosing of autoimmune-prone infant mice with thimerosal-containing vaccines, at the dose given to humans adjusted for mouse weight, resulted in a number of observable effects including growth delay, reduced movement, exaggerated responses, and brain alterations such as increased neuron density and changes in receptors and transporters.
5. Humphrey & Kiningham (2005): after only short (2 hour) exposures, thimerosal at micromolar concentrations caused neuronal membrane damage and alterations leading to cell death.
6. James (2005): the viability of neuronal cell lines was decreased after just 3 hour exposure to 2.5 micromolar concentrations of thimerosal.
7. Makani & Yel (2002) – thimerosal at micromolar amounts causes cell death (apoptosis) in immune cells (T cells).
8. Mutkus & Aschner (2005) – thimerosal alters glutamate transporter function at low micromolar concentrations. Glutamate is a neurotransmitter and is necessary for proper brain functioning.
9. Parran (2005)- thimerosal causes DNA fragmentation of neuronal cells and disrupts neuronal growth factor signaling at micromolar and even nanomolar concentrations.
10. Ueha-Ishibashi (2004: thimerosal at low concentrations is as toxic to rat neurons as methylmercury. The FDA and EPA use methylmercury as their toxicity standard, so demonstration of equivalence shows the potential of thimerosal to cause the same harm as methylmercury, for which more research exists.
11. Waly & Deth (2004): thimerosal inhibits critical DNA methylation and attentional pathways at nanomolar concentrations, leading to alterations in brain function.
12. Westphal (2003) – thimerosal at nanomolar concentrations causes DNA damage in immune cells (lymphocytes) leading to cell death.
1. Baskin DS, Ngo H, Didenko VV.Thimerosal induces DNA breaks, caspase-3 activation, membrane damage, and cell death in cultured human neurons and fibroblasts. Toxicological Sciences. 2003 Aug;74(2):361-8. Epub 2003 May 28.
2. Burbacher TM, Shen DD, Liberato N, Grant KS, Cernichiari E, Clarkson T.Comparison of blood and brain mercury levels in infant monkeys exposed to methylmercury or vaccines containing thimerosal. Environmental Health Perspectives. 2005 Aug;113(8):1015-21.
3. Havarinasab S, Hultman P. Organic mercury compounds and autoimmunity. Autoimmune Rev. 2005 Jun;4(5):270-5. Epub 2005 Jan 5.
4. Hornig M, Chian D, Lipkin WI. Neurotoxic effects of postnatal thimerosal are mouse strain dependent. Molecular Psychiatry. 2004 Sep;9(9):833-45.
5. Humphrey ML, Cole MP, Pendergrass JC, Kiningham KK. Mitochondrial mediated thimerosal-induced apoptosis in a human neuroblastoma cell line (SK-N-SH). Neurotoxicology. 2005 Jun;26(3):407-16.
6. James SJ, Slikker W 3rd, Melnyk S, New E, Pogribna M, Jernigan S. Thimerosal neurotoxicity is associated with glutathione depletion: protection with glutathione precursors. Neurotoxicology. 2005 Jan;26(1):1-8.
7. Makani S, Gollapudi S, Yel L, Chiplunkar S, Gupta S. Biochemical and molecular basis of thimerosal-induced apoptosis in T cells: a major role of mitochondrial pathway.
Genes & Immunity. 2002 Aug;3(5):270-8.
8. Mutkus L, Aschner JL, Syversen T, Shanker G, Sonnewald U, Aschner M. In vitro uptake of glutamate in GLAST- and GLT-1-transfected mutant CHO-K1 cells is inhibited by the ethylmercury-containing preservative thimerosal. Biological Trace Element Research. 2005 Summer;105(1-3):71-86.
9. Parran DK, Barker A, Ehrich M. Effects of thimerosal on NGF signal transduction and cell death in neuroblastoma cells. Toxicological Sciences. 2005 Jul;86(1):132-40. Epub 2005 Apr 20.
10. Ueha-Ishibashi T, Oyama Y, Nakao H, Umebayashi C, Nishizaki Y, Tatsuishi T, Iwase K, Murao K, Seo H. Effect of thimerosal, a preservative in vaccines, on intracellular Ca2+ concentration of rat cerebellar neurons. Toxicology. 2004 Jan 15;195(1):77-84.
11. Waly M, Olteanu H, Banerjee R, Choi SW, Mason JB, Parker BS, Sukumar S, Shim S, Sharma A, Benzecry JM, Power-Charnitsky VA, Deth RC. Activation of methionine synthase by insulin-like growth factor-1 and dopamine: a target for neurodevelopmental toxins and thimerosal. Molecular Psychiatry. 2004 Apr;9(4):358-70.
12. Westphal GA, Asgari S, Schulz TG, Bünger J, Müller M, Hallier E. Thimerosal induces micronuclei in the cytochalasin B block micronucleus test with human lymphocytes. Archives of Toxicology. 2003 Jan; 77(1):50 – 55.
Baskin DS, Ngo H, Didenko VV.Thimerosal induces DNA breaks, caspase-3 activation, membrane damage, and cell death in cultured human neurons and fibroblasts. Toxicological Sciences. 2003 Aug;74(2):361-8. Epub 2003 May 28.
Thimerosal is an organic mercurial compound used as a preservative in biomedical preparations. Little is known about the reactions of human neuronal and skin cells to its micro- and nanomolar concentrations, which can occur after using thimerosal-containing products. A useful combination of fluorescent techniques for the assessment of thimerosal toxicity is introduced. Short-term thimerosal toxicity was investigated in cultured human cerebral cortical neurons and in normal human fibroblasts. Cells were incubated with 125-nM to 250-microM concentrations of thimerosal for 45 min to 24 h. A 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) dye exclusion test was used to identify nonviable cells and terminal transferase-based nick-end labeling (TUNEL) to label DNA damage. Detection of active caspase-3 was performed in live cell cultures using a cell-permeable fluorescent caspase inhibitor. The morphology of fluorescently labeled nuclei was analyzed. After 6 h of incubation, the thimerosal toxicity was observed at 2 microM based on the manual detection of the fluorescent attached cells and at a 1-microM level with the more sensitive GENios Plus Multi-Detection Microplate Reader with Enhanced Fluorescence. The lower limit did not change after 24 h of incubation. Cortical neurons demonstrated higher sensitivity to thimerosal compared to fibroblasts. The first sign of toxicity was an increase in membrane permeability to DAPI after 2 h of incubation with 250 microM thimerosal. A 6-h incubation resulted in failure to exclude DAPI, generation of DNA breaks, caspase-3 activation, and development of morphological signs of apoptosis. We demonstrate that thimerosal in micromolar concentrations rapidly induce membrane and DNA damage and initiate caspase-3-dependent apoptosis in human neurons and fibroblasts. We conclude that a proposed combination of fluorescent techniques can be useful in analyzing the toxicity of thimerosal.
Burbacher TM, Shen DD, Liberato N, Grant KS, Cernichiari E, Clarkson T.Comparison of blood and brain mercury levels in infant monkeys exposed to methylmercury or vaccines containing thimerosal. Environmental Health Perspectives. 2005 Aug;113(8):1015-21.
Thimerosal is a preservative that has been used in manufacturing vaccines since the 1930s. Reports have indicated that infants can receive ethylmercury (in the form of thimerosal) at or above the U.S. Environmental Protection Agency guidelines for methylmercury exposure, depending on the exact vaccinations, schedule, and size of the infant. In this study we compared the systemic disposition and brain distribution of total and inorganic mercury in infant monkeys after thimerosal exposure with those exposed to MeHg. Monkeys were exposed to MeHg (via oral gavage) or vaccines containing thimerosal (via intramuscular injection) at birth and 1, 2, and 3 weeks of age. Total blood Hg levels were determined 2, 4, and 7 days after each exposure. Total and inorganic brain Hg levels were assessed 2, 4, 7, or 28 days after the last exposure. The initial and terminal half-life of Hg in blood after thimerosal exposure was 2.1 and 8.6 days, respectively, which are significantly shorter than the elimination half-life of Hg after MeHg exposure at 21.5 days. Brain concentrations of total Hg were significantly lower by approximately 3-fold for the thimerosal-exposed monkeys when compared with the MeHg infants, whereas the average brain-to-blood concentration ratio was slightly higher for the thimerosal-exposed monkeys (3.5 +/- 0.5 vs. 2.5 +/- 0.3). A higher percentage of the total Hg in the brain was in the form of inorganic Hg for the thimerosal-exposed monkeys (34% vs. 7%). The results indicate that MeHg is not a suitable reference for risk assessment from exposure to thimerosal-derived Hg. Knowledge of the toxicokinetics and developmental toxicity of thimerosal is needed to afford a meaningful assessment of the developmental effects of thimerosal-containing vaccines.
Havarinasab S, Hultman P. Organic mercury compounds and autoimmunity. Autoimmun Rev. 2005 Jun;4(5):270-5. Epub 2005 Jan 5.
Based on in vitro studies and short-term in vivo studies, all mercurials were for a long time considered as prototypic immunosuppressive substances. Recent studies have confirmed that organic mercurials such as methyl mercury (MeHg) and ethyl mercury (EtHg) are much more potent immunosuppressors than inorganic mercury (Hg). However, Hg interacts with the immune system in the presence of a susceptible genotype to cause immunostimulation, antinucleolar antibodies targeting fibrillarin, and systemic immune-complex (IC) deposits, a syndrome called Hg-induced autoimmunity (HgIA). Recent studies in mice with a susceptible genotype has revealed that the immunosuppressive effect of MeHg and EtHg will within 1-3 weeks be superseded by immunostimulation causing an HgIA-like syndrome. At equimolar doses of Hg, MeHg has the weakest immunostimulating, autoimmunogen, and IC-inducing effect, while the effect of thimerosal is similar to that of inorganic mercury. The immunosuppression is caused by the organic mercurials per se. Since they undergo rapid transformation to inorganic Hg, studies are being undertaken to delineate the importance of the organic substances per se and the newly formed inorganic Hg for induction of autoimmunity.
Hornig M, Chian D, Lipkin WI. Neurotoxic effects of postnatal thimerosal are mouse strain dependent. Molecular Psychiatry. 2004 Sep;9(9):833-45.
The developing brain is uniquely susceptible to the neurotoxic hazard posed by mercurials. Host differences in maturation, metabolism, nutrition, sex, and autoimmunity influence outcomes. How population-based variability affects the safety of the ethylmercury-containing vaccine preservative, thimerosal, is unknown. Reported increases in the prevalence of autism, a highly heritable neuropsychiatric condition, are intensifying public focus on environmental exposures such as thimerosal. Immune profiles and family history in autism are frequently consistent with autoimmunity. We hypothesized that autoimmune propensity influences outcomes in mice following thimerosal challenges that mimic routine childhood immunizations. Autoimmune disease-sensitive SJL/J mice showed growth delay; reduced locomotion; exaggerated response to novelty; and densely packed, hyperchromic hippocampal neurons with altered glutamate receptors and transporters. Strains resistant to autoimmunity, C57BL/6J and BALB/cJ, were not susceptible. These findings implicate genetic influences and provide a model for investigating thimerosal-related neurotoxicity.
Humphrey ML, Cole MP, Pendergrass JC, Kiningham KK. Mitochondrial mediated thimerosal-induced apoptosis in a human neuroblastoma cell line (SK-N-SH). Neurotoxicology. 2005 Jun;26(3):407-16.
Environmental exposure to mercurials continues to be a public health issue due to their deleterious effects on immune, renal and neurological function. Recently the safety of thimerosal, an ethyl mercury-containing preservative used in vaccines, has been questioned due to exposure of infants during immunization. Mercurials have been reported to cause apoptosis in cultured neurons; however, the signaling pathways resulting in cell death have not been well characterized. Therefore, the objective of this study was to identify the mode of cell death in an in vitro model of thimerosal-induced neurotoxicity, and more specifically, to elucidate signaling pathways which might serve as pharmacological targets. Within 2 h of thimerosal exposure (5 microM) to the human neuroblastoma cell line, SK-N-SH, morphological changes, including membrane alterations and cell shrinkage, were observed. Cell viability, assessed by measurement of lactate dehydrogenase (LDH) activity in the medium, as well as the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, showed a time- and concentration-dependent decrease in cell survival upon thimerosal exposure. In cells treated for 24 h with thimerosal, fluorescence microscopy indicated cells undergoing both apoptosis and oncosis/necrosis. To identify the apoptotic pathway associated with thimerosal-mediated cell death, we first evaluated the mitochondrial cascade, as both inorganic and organic mercurials have been reported to accumulate in the organelle. Cytochrome c was shown to leak from the mitochondria, followed by caspase 9 cleavage within 8 h of treatment. In addition, poly(ADP-ribose) polymerase (PARP) was cleaved to form a 85 kDa fragment following maximal caspase 3 activation at 24 h. Taken together these findings suggest deleterious effects on the cytoarchitecture by thimerosal and initiation of mitochondrial-mediated apoptosis.
James SJ, Slikker W 3rd, Melnyk S, New E, Pogribna M, Jernigan S. Thimerosal neurotoxicity is associated with glutathione depletion: protection with glutathione precursors. Neurotoxicology. 2005 Jan;26(1):1-8.
Thimerosol is an antiseptic containing 49.5% ethyl mercury that has been used for years as a preservative in many infant vaccines and in flu vaccines. Environmental methyl mercury has been shown to be highly neurotoxic, especially to the developing brain. Because mercury has a high affinity for thiol (sulfhydryl (-SH)) groups, the thiol-containing antioxidant, glutathione (GSH), provides the major intracellular defense against mercury-induced neurotoxicity. Cultured neuroblastoma cells were found to have lower levels of GSH and increased sensitivity to thimerosol toxicity compared to glioblastoma cells that have higher basal levels of intracellular GSH. Thimerosal-induced cytotoxicity was associated with depletion of intracellular GSH in both cell lines. Pretreatment with 100 microM glutathione ethyl ester or N-acetylcysteine (NAC), but not methionine, resulted in a significant increase in intracellular GSH in both cell types. Further, pretreatment of the cells with glutathione ethyl ester or NAC prevented cytotoxicity with exposure to 15 microM Thimerosal. Although Thimerosal has been recently removed from most children's vaccines, it is still present in flu vaccines given to pregnant women, the elderly, and to children in developing countries. The potential protective effect of GSH or NAC against mercury toxicity warrants further research as possible adjunct therapy to individuals still receiving Thimerosal-containing vaccinations.
Makani S, Gollapudi S, Yel L, Chiplunkar S, Gupta S. Biochemical and molecular basis of thimerosal-induced apoptosis in T cells: a major role of mitochondrial pathway. Genes Immun. 2002 Aug;3(5):270-8.
The major source of thimerosal (ethyl mercury thiosalicylate) exposure is childhood vaccines. It is believed that the children are exposed to significant accumulative dosage of thimerosal during the first 2 years of life via immunization. Because of health-related concerns for exposure to mercury, we examined the effects of thimerosal on the biochemical and molecular steps of mitochondrial pathway of apoptosis in Jurkat T cells. Thimerosal and not thiosalcylic acid (non-mercury component of thimerosal), in a concentration-dependent manner, induced apoptosis in T cells as determined by TUNEL and propidium iodide assays, suggesting a role of mercury in T cell apoptosis. Apoptosis was associated with depolarization of mitochondrial membrane, release of cytochrome c and apoptosis inducing factor (AIF) from the mitochondria, and activation of caspase-9 and caspase-3, but not of caspase-8. In addition, thimerosal in a concentration-dependent manner inhibited the expression of XIAP, cIAP-1 but did not influence cIAP-2 expression. Furthermore, thimerosal enhanced intracellular reactive oxygen species and reduced intracellular glutathione (GSH). Finally, exogenous glutathione protected T cells from thimerosal-induced apoptosis by upregulation of XIAP and cIAP1 and by inhibiting activation of both caspase-9 and caspase-3. These data suggest that thimerosal induces apoptosis in T cells via mitochondrial pathway by inducing oxidative stress and depletion of GSH.
Mutkus L, Aschner JL, Syversen T, Shanker G, Sonnewald U, Aschner M. In vitro uptake of glutamate in GLAST- and GLT-1-transfected mutant CHO-K1 cells is inhibited by the ethylmercury-containing preservative thimerosal. Biological Trace Element Research. 2005 Summer;105(1-3):71-86.
Thimerosal, also known as thimersal, Merthrolate, or sodiumethyl-mercurithiosalicylate, is an organic mercurial compound that is used in a variety of commercial as well as biomedical applications. As a preservative, it is used in a number of vaccines and pharmaceutical products. Its active ingredient is ethylmercury. Both inorganic and organic mercurials are known to interfere with glutamate homeostasis. Brain glutamate is removed mainly by astrocytes from the extracellular fluid via high-affinity astroglial Na+-dependent excitatory amino acid transporters, glutamate/ aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1). The effects of thimerosal on glutamate homeostasis have yet to be determined. As a first step in this process, we examined the effects of thimerosal on the transport of [3H]-d-aspartate, a nonmetabolizable glutamate analog, in Chinese hamster ovary (CHO) cells transfected with two glutamate transporter subtypes, GLAST (EAAT1) and GLT-1 (EAAT2). Additionally, studies were undertaken to determine the effects of thimerosal on mRNA and protein levels of these transporters. The results indicate that thimerosal treatment caused significant but selective changes in both glutamate transporter mRNA and protein expression in CHO cells. Thimerosal-mediated inhibition of glutamate transport in the CHO-K1 cell line DdB7 was more pronounced in the GLT-1-transfected cells compared with the GLAST- transfected cells. These studies suggest that thimerosal accumulation in the central nervous system might contribute to dysregulation of glutamate homeostasis.
Parran DK, Barker A, Ehrich M. Effects of thimerosal on NGF signal transduction and cell death in neuroblastoma cells. Toxicological Sciences. 2005 Jul;86(1):132-40. Epub 2005 Apr 20.
Signaling through neurotrophic receptors is necessary for differentiation and survival of the developing nervous system. The present study examined the effects of the organic mercury compound thimerosal on nerve growth factor signal transduction and cell death in a human neuroblastoma cell line (SH-SY5Y cells). Following exposure to 100 ng/ml NGF and increasing concentrations of thimerosal (1 nM-10 microM), we measured the activation of TrkA, MAPK, and PKC-delta. In controls, the activation of TrkA MAPK and PKC-delta peaked after 5 min of exposure to NGF and then decreased but was still detectable at 60 min. Concurrent exposure to increasing concentrations of thimerosal and NGF for 5 min resulted in a concentration-dependent decrease in TrkA and MAPK phosphorylation, which was evident at 50 nM for TrkA and 100 nM for MAPK. Cell viability was assessed by the LDH assay. Following 24-h exposure to increasing concentrations of thimerosal, the EC50 for cell death in the presence or absence of NGF was 596 nM and 38.7 nM, respectively. Following 48-h exposure to increasing concentrations of thimerosal, the EC50 for cell death in the presence and absence of NGF was 105 nM and 4.35 nM, respectively. This suggests that NGF provides protection against thimerosal cytotoxicity. To determine if apoptotic versus necrotic cell death was occurring, oligonucleosomal fragmented DNA was quantified by ELISA. Control levels of fragmented DNA were similar in both the presence and absence of NGF. With and without NGF, thimerosal caused elevated levels of fragmented DNA appearing at 0.01 microM (apoptosis) to decrease at concentrations >1 microM (necrosis). These data demonstrate that thimerosal could alter NGF-induced signaling in neurotrophin-treated cells at concentrations lower than those responsible for cell death.
Ueha-Ishibashi T, Oyama Y, Nakao H, Umebayashi C, Nishizaki Y, Tatsuishi T, Iwase K, Murao K, Seo H. Effect of thimerosal, a preservative in vaccines, on intracellular Ca2+ concentration of rat cerebellar neurons. Toxicology. 2004 Jan 15;195(1):77-84.
The effect of thimerosal, an organomercurial preservative in vaccines, on cerebellar neurons dissociated from 2-week-old rats was compared with those of methylmercury using a flow cytometer with appropriate fluorescent dyes. Thimerosal and methylmercury at concentrations ranging from 0.3 to 10 microM increased the intracellular concentration of Ca2+ ([Ca2+]i) in a concentration-dependent manner. The potency of 10 microM thimerosal to increase the [Ca2+]i was less than that of 10 microM methylmercury. Their effects on the [Ca2+]i were greatly attenuated, but not completely suppressed, under external Ca(2+)-free condition, suggesting a possibility that both agents increase membrane Ca2+ permeability and release Ca2+ from intracellular calcium stores. The effect of 10 microM thimerosal was not affected by simultaneous application of 30 microM L-cysteine whereas that of 10 microM methylmercury was significantly suppressed. The potency of thimerosal was similar to that of methylmercury in the presence of L-cysteine. Both agents at 1 microM or more similarly decreased the cellular content of glutathione in a concentration-dependent manner, suggesting an increase in oxidative stress. Results indicate that thimerosal exerts some cytotoxic actions on cerebellar granule neurons dissociated from 2-week-old rats and its potency is almost similar to that of methylmercury.
Waly M, Olteanu H, Banerjee R, Choi SW, Mason JB, Parker BS, Sukumar S, Shim S, Sharma A, Benzecry JM, Power-Charnitsky VA, Deth RC. Activation of methionine synthase by insulin-like growth factor-1 and dopamine: a target for neurodevelopmental toxins and thimerosal. Molecular Psychiatry. 2004 Apr;9(4):358-70.
Methylation events play a critical role in the ability of growth factors to promote normal development. Neurodevelopmental toxins, such as ethanol and heavy metals, interrupt growth factor signaling, raising the possibility that they might exert adverse effects on methylation. We found that insulin-like growth factor-1 (IGF-1)- and dopamine-stimulated methionine synthase (MS) activity and folate-dependent methylation of phospholipids in SH-SY5Y human neuroblastoma cells, via a PI3-kinase- and MAP-kinase-dependent mechanism. The stimulation of this pathway increased DNA methylation, while its inhibition increased methylation-sensitive gene expression. Ethanol potently interfered with IGF-1 activation of MS and blocked its effect on DNA methylation, whereas it did not inhibit the effects of dopamine. Metal ions potently affected IGF-1 and dopamine-stimulated MS activity, as well as folate-dependent phospholipid methylation: Cu(2+) promoted enzyme activity and methylation, while Cu(+), Pb(2+), Hg(2+) and Al(3+) were inhibitory. The ethylmercury-containing preservative thimerosal inhibited both IGF-1- and dopamine-stimulated methylation with an IC(50) of 1 nM and eliminated MS activity. Our findings outline a novel growth factor signaling pathway that regulates MS activity and thereby modulates methylation reactions, including DNA methylation. The potent inhibition of this pathway by ethanol, lead, mercury, aluminum and thimerosal suggests that it may be an important target of neurodevelopmental toxins.
Götz A. Westphal, Soha Asgari, Thomas G. Schulz, Jürgen Bünger, Michael Müller, Ernst Hallier Thimerosal induces micronuclei in the cytochalasin B block micronucleus test with human lymphocytes. Archives of Toxicology. 2003 Jan; 77(1):50 – 55.
Thimerosal is a widely used preservative in health care products, especially in vaccines. Due to possible adverse health effects, investigations on its metabolism and toxicity are urgently needed. An in vivo study on chronic toxicity of thimerosal in rats was inconclusive and reports on genotoxic effects in various in vitro systems were contradictory. Therefore, we reinvestigated thimerosal in the cytochalasin B block micronucleus test. Glutathione S-transferases were proposed to be involved in the detoxification of thimerosal or its decomposition products. Since the outcome of genotoxicity studies can be dependent on the metabolic competence of the cells used, we were additionally interested whether polymorphisms of glutathione S-transferases (GSTM1, GSTT1, or GSTP1) may influence the results of the micronucleus test with primary human lymphocytes. Blood samples of six healthy donors of different glutathione S-transferase genotypes were included in the study. At least two independent experiments were performed for each blood donor. Significant induction of micronuclei was seen at concentrations between 0.05-0.5 µg/ml in 14 out of 16 experiments. Thus, genotoxic effects were seen even at concentrations which can occur at the injection site. Toxicity and toxicity-related elevation of micronuclei was seen at and above 0.6 µg/ml thimerosal. Marked individual and intraindividual variations in the in vitro response to thimerosal among the different blood donors occurred. However, there was no association observed with any of the glutathione S-transferase polymorphism investigated. In conclusion, thimerosal is genotoxic in the cytochalasin B block micronucleus test with human lymphocytes. These data raise some concern on the widespread use of thimerosal.