Teresa Binstock
Researcher in Developmental & Behavioral Neuroanatomy
May 23, 2009
A newly published study
reports that "Early-life exposure to air pollution from indoor gas
appliances may be negatively associated with neuropsychological
development through the first 4 years of life, particularly among
genetically susceptible children." (1) Indeed and related to autism,
adverse effects were more likely if the child has a particular allele
related to glutathione (GSTP1). As of May 2009, the study is not yet
free online but a synopsis has been posted by
EnvironmentalHealthNews.org (2).
The new findings about GSTP1 are consistent with previous studies
wherein glutathione and related alleles have been implicated in autism.
Williams and colleagues reported that "Overtransmission of the GSTP1*A
haplotype to case mothers suggests that action in the mother during
pregnancy likely increases the likelihood of AD [autistic disorder] in
her fetus." (3). Jill James and her team have published several studies
implicating glutathione irregularities and related genes in autistic
children and their families (eg, 4-5) and have reported clinical
benefit when these impaired pathways are addressed nutritionally (eg,
6). Importantly, children with weak alleles related to glutathione are
likely to have difficulty detoxifying vaccine ingredients such as
thimerosal and aluminum (eg, 7-8), which are implicated in
neurotoxicity and DNA damage (genotoxicity) (eg, 9-12). Furthermore,
thimerosal itself impairs a glutathione-related enzyme, thus augmenting
an infant's or toddler's impaired detoxification of numerous pollutants
(13), especially in children having a weak allele in metabolic pathways
related to glutathione.
For children and mothers having one or more weak allele in glutathione
pathways, we call attention to findings that thimerosal adversely
affects mitochondria (eg, 14-15) and calcium pathways (eg, 16).
When prominent individuals such as Bernadine Healy, M.D., former NIH
director, and Duane Alexander, M.D., director of the NICHD, and
vaccinologist Gregory Poland, M.D., state that better studies and safer
vaccines are needed (eg, 17-19), we are well served to pay attention,
because a growing body of peer-reviewed findings are elucidating
alleles that predispose to damage from aluminum, thimerosal, household
gas, and other pollutants.
References:
1. Association of Early-life Exposure to Household Gas Appliances and
Indoor Nitrogen Dioxide With Cognition and Attention Behavior in
Preschoolers
Morales E et al.
American Journal of Epidemiology Advance Access
http://aje.oxfordjournals.org/cgi/content/abstract/kwp067v1
http://aje.oxfordjournals.org/cgi/reprint/kwp067v1
The authors investigated the association of early-life exposure to
indoor air pollution with neuropsychological development in
preschoolers and assessed whether this association differs by
glutathione-S-transferase gene (GSTP1) polymorphisms. A prospective,
population-based birth cohort was set up in Menorca, Spain, in
1997–1999 (n = 482). Children were assessed for cognitive functioning
(McCarthy Scales of Children's Abilities) and attention-hyperactivity
behaviors (Diagnostic and Statistical Manual of Mental Disorders, 4th
Edition) at age 4 years. During the first 3 months of life, information
about gas appliances at home and indoor nitrogen dioxide concentration
was collected at each participant's home (n = 398, 83%). Genotyping was
conducted for the GSTP1 coding variant Ile105Val. Use of gas appliances
was inversely associated with cognitive outcomes (β coefficient
for general cognition = –5.10, 95% confidence interval (CI): –9.92,
–0.28; odds ratio for inattention symptoms = 3.59, 95% CI: 1.14,
11.33), independent of social class and other confounders. Nitrogen
dioxide concentrations were associated with cognitive function (a
decrease of 0.27 point per 1 ppb, 95% CI: –0.48, –0.07) and inattention
symptoms (odds ratio = 1.06, 95% CI: 1.01, 1.12). The deleterious
effect of indoor pollution from gas appliances on neuropsychological
outcomes was stronger in children with the GSTP1 Val-105 allele.
Early-life exposure to air pollution from indoor gas appliances may be
negatively associated with neuropsychological development through the
first 4 years of life, particularly among genetically susceptible
children.
2. Cognition, attention altered in youngsters who live with gas
appliances.
http://www.environmentalhealthnews.org/ehs/newscience/gas-appliances-linked-to-lower-cognition-and-attention/
Preschoolers who lived in homes using gas appliances scored lower on
cognitive tests and had a higher likelihood of exhibiting inattention
behaviors than those in homes without gas appliances, finds a recent
study. The effects on memory, verbal skills and the coordination of
complex behaviors were greater when more gas appliances were used in
the homes. They were also more pronounced in children with a certain
gene type involved with the detoxification of toxic exposures.
3. Risk of autistic disorder in affected offspring of mothers with a
glutathione S-transferase P1 haplotype.
Williams TA et al.
Arch Pediatr Adolesc Med. 2007 Apr;161(4):356-61.
Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.
http://archpedi.ama-assn.org/cgi/content/full/161/4/356
OBJECTIVE: To test whether polymorphisms of the glutathione
S-transferase P1 gene (GSTP1) act in the mother during pregnancy to
contribute to the phenotype of autistic disorder (AD) in her fetus.
DESIGN: Transmission disequilibrium testing (TDT) in case mothers and
maternal grandparents. SETTING: Autistic disorder may result from
multiple genes and environmental factors acting during pregnancy and
afterward. Teratogenic alleles act in mothers during pregnancy to
contribute to neurodevelopmental disorders in their offspring; however,
only a handful have been identified. GSTP1 is a candidate
susceptibility gene for AD because of its tissue distribution and its
role in oxidative stress, xenobiotic metabolism, and JNK regulation.
PARTICIPANTS: We genotyped GSTP1*G313A and GSTP1*C341T polymorphisms in
137 members of 49 families with AD. All probands received a clinical
diagnosis of AD by Autism Diagnostic Interview-Revised and Autism
Diagnostic Observation Schedule-Generic testing. MAIN OUTCOME MEASURES:
Association of haplotypes with AD was tested by the TDT-Phase program,
using the expectation-maximization (EM) algorithm for uncertain
haplotypes and for incomplete parental genotypes, with standard
measures of statistical significance. RESULTS: The GSTP1*A haplotype
was overtransmitted to case mothers (P = .01 [P = .03 using permutation
testing]; odds ratio, 2.67 [95% confidence interval, 1.39-5.13]).
Results of the combined haplotype and genotype analyses suggest that
the GSTP1-313 genotype alone determined the observed haplotype effect.
CONCLUSIONS: Overtransmission of the GSTP1*A haplotype to case mothers
suggests that action in the mother during pregnancy likely increases
the likelihood of AD in her fetus. If this is confirmed and is a result
of a gene-environment interaction occurring during pregnancy, these
findings could lead to the design of strategies for prevention or
treatment.
4. Metabolic biomarkers of increased oxidative stress and impaired
methylation capacity in children with autism.
James SJ et al.
University of Arkansas for Medical Sciences
Am J Clin Nutr. 2004 Dec;80(6):1611-7.
http://www.ajcn.org/cgi/content/full/80/6/1611
BACKGROUND: Autism is a complex neurodevelopmental disorder that
usually presents in early childhood and that is thought to be
influenced by genetic and environmental factors. Although abnormal
metabolism of methionine and homocysteine has been associated with
other neurologic diseases, these pathways have not been evaluated in
persons with autism. OBJECTIVE: The purpose of this study was to
evaluate plasma concentrations of metabolites in the methionine
transmethylation and transsulfuration pathways in children diagnosed
with autism. DESIGN: Plasma concentrations of methionine,
S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), adenosine,
homocysteine, cystathionine, cysteine, and oxidized and reduced
glutathione were measured in 20 children with autism and in 33 control
children. On the basis of the abnormal metabolic profile, a targeted
nutritional intervention trial with folinic acid, betaine, and
methylcobalamin was initiated in a subset of the autistic children.
RESULTS: Relative to the control children, the children with autism had
significantly lower baseline plasma concentrations of methionine, SAM,
homocysteine, cystathionine, cysteine, and total glutathione and
significantly higher concentrations of SAH, adenosine, and oxidized
glutathione. This metabolic profile is consistent with impaired
capacity for methylation (significantly lower ratio of SAM to SAH) and
increased oxidative stress (significantly lower redox ratio of reduced
glutathione to oxidized glutathione) in children with autism. The
intervention trial was effective in normalizing the metabolic imbalance
in the autistic children. CONCLUSIONS: An increased vulnerability to
oxidative stress and a decreased capacity for methylation may
contribute to the development and clinical manifestation of autism.
5. Metabolic endophenotype and related genotypes are associated with
oxidative stress in children with autism.
James SJ et al.
Am J Med Genet B Neuropsychiatr Genet. 2006 Dec 5;141B(8):947-56.
http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=2610366&blobtype=pdf
Autism is a behaviorally defined neurodevelopmental disorder usually
diagnosed in early childhood that is characterized by impairment in
reciprocal communication and speech, repetitive behaviors, and social
withdrawal. Although both genetic and environmental factors are thought
to be involved, none have been reproducibly identified. The metabolic
phenotype of an individual reflects the influence of endogenous and
exogenous factors on genotype. As such, it provides a window through
which the interactive impact of genes and environment may be viewed and
relevant susceptibility factors identified. Although abnormal
methionine metabolism has been associated with other neurologic
disorders, these pathways and related polymorphisms have not been
evaluated in autistic children. Plasma levels of metabolites in
methionine transmethylation and transsulfuration pathways were measured
in 80 autistic and 73 control children. In addition, common polymorphic
variants known to modulate these metabolic pathways were evaluated in
360 autistic children and 205 controls. The metabolic results indicated
that plasma methionine and the ratio of S-adenosylmethionine (SAM) to
S-adenosylhomocysteine (SAH), an indicator of methylation capacity,
were significantly decreased in the autistic children relative to
age-matched controls. In addition, plasma levels of cysteine,
glutathione, and the ratio of reduced to oxidized glutathione, an
indication of antioxidant capacity and redox homeostasis, were
significantly decreased. Differences in allele frequency and/or
significant gene-gene interactions were found for relevant genes
encoding the reduced folate carrier (RFC 80G > A), transcobalamin II
(TCN2 776G > C), catechol-O-methyltransferase (COMT 472G > A),
methylenetetrahydrofolate reductase (MTHFR 677C > T and 1298A >
C), and glutathione-S-transferase (GST M1). We propose that an
increased vulnerability to oxidative stress (endogenous or
environmental) may contribute to the development and clinical
manifestations of autism.
6. Efficacy of methylcobalamin and folinic acid treatment on
glutathione redox status in children with autism.
James SJ et al.
Am J Clin Nutr. 2009 Jan;89(1):425-30. Epub 2008 Dec 3.
http://www.ajcn.org/cgi/content/full/89/1/425
BACKGROUND: Metabolic abnormalities and targeted treatment trials have
been reported for several neurobehavioral disorders but are relatively
understudied in autism. OBJECTIVE: The objective of this study was to
determine whether or not treatment with the metabolic precursors,
methylcobalamin and folinic acid, would improve plasma concentrations
of transmethylation/transsulfuration metabolites and glutathione redox
status in autistic children. DESIGN: In an open-label trial, 40
autistic children were treated with 75 microg/kg methylcobalamin (2
times/wk) and 400 microg folinic acid (2 times/d) for 3 mo. Metabolites
in the transmethylation/transsulfuration pathway were measured before
and after treatment and compared with values measured in age-matched
control children. RESULTS: The results indicated that pretreatment
metabolite concentrations in autistic children were significantly
different from values in the control children. The 3-mo intervention
resulted in significant increases in cysteine, cysteinylglycine, and
glutathione concentrations (P < 0.001). The oxidized disulfide form
of glutathione was decreased and the glutathione redox ratio increased
after treatment (P < 0.008). Although mean metabolite concentrations
were improved significantly after intervention, they remained below
those in unaffected control children. CONCLUSION: The significant
improvements observed in transmethylation metabolites and glutathione
redox status after treatment suggest that targeted nutritional
intervention with methylcobalamin and folinic acid may be of clinical
benefit in some children who have autism. This trial was registered at
(clinicaltrials.gov) as NCT00692315.
7. Cellular and mitochondrial glutathione redox imbalance in
lymphoblastoid cells derived from children with autism.
James SJ et al.
FASEB J. 2009 Mar 23. [Epub ahead of print]
http://www.fasebj.org/cgi/rapidpdf/fj.08-128926v1
Research into the metabolic phenotype of autism has been relatively
unexplored despite the fact that metabolic abnormalities have been
implicated in the pathophysiology of several other neurobehavioral
disorders. Plasma biomarkers of oxidative stress have been reported in
autistic children; however, intracellular redox status has not yet been
evaluated. Lymphoblastoid cells (LCLs) derived from autistic children
and unaffected controls were used to assess relative concentrations of
reduced glutathione (GSH) and oxidized disulfide glutathione (GSSG) in
cell extracts and isolated mitochondria as a measure of intracellular
redox capacity. The results indicated that the GSH/GSSG redox ratio was
decreased and percentage oxidized glutathione increased in both cytosol
and mitochondria in the autism LCLs. Exposure to oxidative stress via
the sulfhydryl reagent thimerosal resulted in a greater decrease in the
GSH/GSSG ratio and increase in free radical generation in autism
compared to control cells. Acute exposure to physiological levels of
nitric oxide decreased mitochondrial membrane potential to a greater
extent in the autism LCLs, although GSH/GSSG and ATP concentrations
were similarly decreased in both cell lines. These results suggest that
the autism LCLs exhibit a reduced glutathione reserve capacity in both
cytosol and mitochondria that may compromise antioxidant defense and
detoxification capacity under prooxidant conditions.
8. Homozygous gene deletions of the glutathione S-transferases M1 and
T1 are associated with thimerosal sensitization.
Westphal GA et al.
Georg-August-Universität Göttingen
Int Arch Occup Environ Health. 2000 Aug;73(6):384-8.
OBJECTIVE: Thimerosal is an important preservative in vaccines and
ophthalmologic preparations. The substance is known to be a type IV
sensitizing agent. High sensitization rates were observed in
contact-allergic patients and in health care workers who had been
exposed to thimerosal-preserved vaccines. There is evidence for the
involvement of the glutathione system in the metabolism of thimerosal
or its decomposition products (organomercury alkyl compounds). Thus
detoxification by polymorphically expressed glutathione S-transferases
such as GSTT1 and GSTM1 might have a protective effect against
sensitization by these substances. METHODS: To address this question, a
case control study was conducted, including 91 Central European
individuals with a positive patch-test reaction to thimerosal. This
population was compared with 169 healthy controls and additionally with
114 individuals affected by an allergy against para-substituted aryl
compounds. The latter population was included in order to test whether
possible associations were due to substance-specific effects, or were a
general feature connected with type IV immunological diseases.
Homozygous deletions of GSTT1 and GSTM1 were determined by polymerase
chain reaction. RESULTS: Glutathione S-transferase M1 deficiency was
significantly more frequent among patients sensitized to thimerosal
(65.9%, P = 0.013) compared with the healthy control group (49.1%) and
the "para-compound" group (48%, P = 0.034). Glutathione S-transferase
T1 deficiency in the thimerosal/mercury group (19.8%) was barely
elevated versus healthy controls (16.0%) and the "para-compound" group
(14.0%). The combined deletion (GSTT1-/GSTM1-) was markedly more
frequent among thimerosal-sensitized patients than in healthy controls
(17.6% vs. 6.5%, P = 0.0093) and in the "para-compound" group (17.6%
vs. 6.1%, P =0.014), revealing a synergistic effect of these enzyme
deficiencies (healthy controls vs. thimerosal GSTM1 negative
individuals, OR = 2.0 [CI = 1.2-3.4], GSTT1-, OR = 1.2 [CI = 0.70-2.1],
GSTM1/T1-, OR = 3.1 [CI = 1.4-6.5]). CONCLUSIONS: Since the
glutathione-dependent system was repeatedly shown to be involved in the
metabolism of thimerosal decomposition products, the observed
association may be of functional relevance.
9. Thimerosal induces micronuclei in the cytochalasin B block
micronucleus test with human lymphocytes.
Westphal GA et al.
Arch Toxicol. 2003 Jan;77(1):50-5. Epub 2002 Nov 6.
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 micro g/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 micro g/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.
10. Thimerosal neurotoxicity is associated with glutathione depletion:
protection with glutathione precursors.
James SJ et al.
Neurotoxicology. 2005 Jan;26(1):1-8.
http://linkinghub.elsevier.com/retrieve/pii/S0161-813X(04)00114-7
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.
11. Thimerosal induces DNA breaks, caspase-3 activation, membrane
damage, and cell death in cultured human neurons and fibroblasts.
Baskin DS, Ngo H, Didenko VV.
Baylor College of Medicine
Toxicol Sci. 2003 Aug;74(2):361-8. Epub 2003 May 28.
http://toxsci.oxfordjournals.org/cgi/content/full/74/2/361
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.
12. Glutathione depletion promotes aluminum-mediated cell death of PC12
cells.
Satoh E et al.
Faculty of Pharmaceutical Sciences, Hokuriku University
Biol Pharm Bull. 2005 Jun;28(6):941-6.
http://www.jstage.jst.go.jp/article/bpb/28/6/28_941/_article
Exposure of rat phenochromocytoma cells (PC12 cells) to aluminum
maltolate complex, Al(maltol)3, induced a decrease in intracellular
glutathione (GSH) concentration, resulting in a facilitated release of
lactate dehydrogenase (LDH) from the cell and an increase in trypan
blue-stained cells. Similar phenomena were observed as the cells were
treated with L-buthione-[S,R]-sulfoximine (BSO) in the presence of
Al(maltol)3. On the other hand, treatment of PC 12 cells with BSO alone
in the absence of Al(maltol)3 did not affect the cell viability.
Pre-treatment of PC12 cells with N-acetylcysteine (NAC) for 30 min
before a 48 h-exposure to Al(maltol)3 effectively protected the cells
from Al(maltol)3 toxicity by increasing intracellular GSH
concentration. NAC also effectively inhibited reactive oxygen species
(ROS) generation induced by treatment of the cells with Al(maltol)3.
However, several lipophilic radical scavengers such as alpha-tocopherol
and 3(2)-tert-butyl-4-hydroxyanisole, and an iron chelator,
desferrioxamine, did not prevent Al(maltol)3-mediated ROS production or
the decrease of cell viability. Based on these results, we discussed
the role of intracellular GSH against the onset of aluminum toxicity in
the context of ROS production.
13. Inhibition of the human erythrocytic glutathione-S-transferase T1
(GST T1) by thimerosal.
Müller M et al.
Int J Hyg Environ Health. 2001 Jul;203(5-6):479-81.
We have investigated the interaction of thimerosal, a widely used
antiseptic and preservative, with the human erythrocytic GST T1
(glutathione-S-transferase T1). This detoxifying enzyme is expressed in
the erythrocytes of solely the human species and it displays a genetic
polymorphism. Due to this polymorphism about 25% of the individuals of
the caucasian population lack this activity ("non-conjugators"), while
75% show it ("conjugators") (Hallier, E., et al., 1993). Using our
newly developed HPLC-fluorescence detection assay (Müller, M., et al.,
2001) we have profiled the kinetics of enzyme inhibition in erythrocyte
lysates of two individuals previously identified as "normal conjugator"
(medium enzyme activity) and "super-conjugator" (very high activity).
For the normal conjugator we have determined a 2.77 mM thimerosal
concentration to inhibit 50% of the GST T1 activity. In the case of the
super-conjugator a 2.3 mM thimerosal concentration causes a 50%
inhibition of the enzyme activity. For both phenotypes a 14.8 mM
thimerosal concentration results in residual enzyme activities equal to
those typically detected in non-conjugator lysates. Thus, sufficiently
high doses of thimerosal may be able to change the phenotypic status of
an individual--at least in vitro--by inhibition of the GST T1
enzyme.
14. Thimerosal induces neuronal cell apoptosis by causing cytochrome c
and apoptosis-inducing factor release from mitochondria.
Yel L et al.
Department of Medicine, University of California
Int J Mol Med. 2005 Dec;16(6):971-7.
There is a worldwide increasing concern over the neurological risks of
thimerosal (ethylmercury thiosalicylate) which is an organic mercury
compound that is commonly used as an antimicrobial preservative. In
this study, we show that thimerosal, at nanomolar concentrations,
induces neuronal cell death through the mitochondrial pathway.
Thimerosal, in a concentration- and time-dependent manner, decreased
cell viability as assessed by calcein-ethidium staining and caused
apoptosis detected by Hoechst 33258 dye. Thimerosal-induced apoptosis
was associated with depolarization of mitochondrial membrane,
generation of reactive oxygen species, and release of cytochrome c and
apoptosis-inducing factor (AIF) from mitochondria to cytosol. Although
thimerosal did not affect cellular expression of Bax at the protein
level, we observed translocation of Bax from cytosol to mitochondria.
Finally, caspase-9 and caspase-3 were activated in the absence of
caspase-8 activation. Our data suggest that thimerosal causes apoptosis
in neuroblastoma cells by changing the mitochondrial
microenvironment.
15. Mitochondrial mediated thimerosal-induced apoptosis in a human
neuroblastoma cell line (SK-N-SH).
Humphrey ML et al.
Joan C. Edwards School of Medicine, Marshall University, Huntington, WV
25704-9388, USA.
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.
16. Uncoupling of ATP-mediated calcium signaling and dysregulated
interleukin-6 secretion in dendritic cells by nanomolar thimerosal.
Goth SR et al.
University of California-Davis, Davis, California, USA.
Environ Health Perspect. 2006 Jul;114(7):1083-91.
http://www.ehponline.org/members/2006/8881/8881.html
Dendritic cells (DCs) , a rare cell type widely distributed in the
soma, are potent antigen-presenting cells that initiate primary immune
responses. DCs rely on intracellular redox state and calcium (Ca2+)
signals for proper development and function, but the relationship
between these two signaling systems is unclear. Thimerosal (THI) is a
mercurial used to preserve vaccines and consumer products, and is used
experimentally to induce Ca2+ release from microsomal stores. We tested
adenosine triphosphate (ATP) -mediated Ca2+ responses of DCs
transiently exposed to nanomolar THI. Transcriptional and
immunocytochemical analyses show that murine myeloid immature DCs
(IDCs) and mature DCs (MDCs) express inositol 1,4,5-trisphosphate
receptor (IP3R) and ryanodine receptor (RyR) Ca2+ channels, known
targets of THI. IDCs express the RyR1 isoform in a punctate
distribution that is densest near plasma membranes and within dendritic
processes, whereas IP3Rs are more generally distributed. RyR1
positively and negatively regulates purinergic signaling because
ryanodine (Ry) blockade a) recruited 80% more ATP responders, b)
shortened ATP-mediated Ca2+ transients > 2-fold, and c) produced a
delayed and persistent rise (>/= 2-fold) in baseline Ca2+. THI (100
nM, 5 min) recruited more ATP responders, shortened the ATP-mediated
Ca2+ transient (>/= 1.4-fold) , and produced a delayed rise (>/=
3-fold) in the Ca2+ baseline, mimicking Ry. THI and Ry, in combination,
produced additive effects leading to uncoupling of IP3R and RyR1
signals. THI altered ATP-mediated interleukin-6 secretion, initially
enhancing the rate of cytokine secretion but suppressing cytokine
secretion overall in DCs.DCs are exquisitely sensitive to THI, with one
mechanism involving the uncoupling of positive and negative regulation
of Ca2+ signals contributed by RyR1.
17. Fighting the Autism-Vaccine War
By Bernadine Healy, M.D. [former director of NIH]
http://health.usnews.com/articles/health/brain-and-behavior/2008/04/10/fighting-the-autism-vaccine-war.html
18. Duane Alexander. M.D., NICHD director.
Quoted in: NIH Agency Head: Vaccine-Autism Research is "Legitimate"
- by David Kirby
http://www.huffingtonpost.com/david-kirby/nih-agency-head-vaccine-a_b_170034.html
19. Adversomics: the emerging field of vaccine adverse event
immunogenetics.
Poland GA et al.
Pediatr Infect Dis J. 2009 May;28(5):431-2.
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