Opposition to Anti-Thimerosal Legislation - Please forward

[Guest guest]

<http://rs6.net/on.jsp?t=1101265646043.0.1011367688197.1 & o=http://ui.constan

tcontact.com/images/p1x1.gif>

<http://www.themercurygenerationmarch.org/images/MGMLogo.gif>

Opposition to Anti-Thimerosal Legislation

We have just received a

<http://rs6.net/tn.jsp?t=8omyiubab.0.c5fyiubab.faomd6n6.1 & p=http%3A%2F%2Fwww

..nationalautismassociation.org%2FVaccines%2520and%2520thimerosal%2520letter.

pdf> 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

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 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

Mcs. 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.

RESEARCH SUMMARY

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. (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.

REFERENCES

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,

son 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. 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, 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.

RESEARCH ABSTRACTS

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, son

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.

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, 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, G. Schulz, Jürgen Bünger,

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.

<http://rs6.net/tn.jsp?t=8omyiubab.0.c6s4ctbab.faomd6n6.1 & p=http%3A%2F%2Fwww

..themercurygenerationmarch.org%2F> Click here to visit our website