: Opposition to Anti-Thimerosal Legislation

[Guest guest]

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

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.

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.

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.

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.

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?

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.

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.

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