Download Vaccines and Autism - Vanderbilt Kennedy Center

Metals in ASD
Michael Aschner
Dept of Pediatrics & Pharmacology,
& the Kennedy Center for Research on Human Development,
Vanderbilt University Medical Center, Nashville, TN
Outline
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Vaccines
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Methylmercury vs ethylmercury (thimerosal)
Other metals in ASD
 General considerations on the validity of the
studies – research needs
 Polymorphisms in “metal handling” genes
and ASD
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Thimerosal-Containing Vaccines and
Autistic Spectrum Disorder
Parker et al., Pediatrics 114:793-804, 2004
Vaccines and Autism
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Two studies performed in the UK examined
whether thimerosal in vaccines caused
neurodevelopmental or psychological problems
(Parker et al., Pediatrics, 2004);
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neither found evidence that early exposure to
thimerosal was harmful.
Thompson et al. (NEJM, 2007), also found no
evidence of neurologic problems in children
exposed to mercury-containing vaccines or
immune globulins.
Mercury and neuropsychological
outcomes
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Enrolled 1047 children (ages 7 and 10)
Administered standardized tests assessing 42
neuropsychological outcomes.
Exposure to Hg from thimerosal was determined
from computerized immunization, medical, personal
immunization records, and parent interviews.
Association between current neuropsychological
performance and exposure to mercury during the
prenatal period, the neonatal period (birth to 28
days), and the first 7 months of life.
Thompson et al., NEJM, 2007
Mercury and neuropsychological
outcomes
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Among the 42 neuropsychological outcomes, a
few significant associations detected with
exposure to Hg.
Small and almost equally divided between
positive and negative effects.
Higher prenatal Hg exposure was associated
with better performance on one measure of
language and poorer performance on one
measure of attention and executive functioning.
Increasing levels of Hg exposure from birth to 7
months were associated with better performance
on one measure of fine motor coordination and
on one measure of attention and executive
functioning.
Thompson et al., NEJM, 2007
Mercury and neuropsychological
outcomes
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Increasing mercury exposure from birth to 28
days was associated with poorer performance
on one measure of speech articulation and
better performance on one measure of fine
motor coordination.
Study does not support a causal association
between early exposure to Hg from thimerosalcontaining vaccines and immune globulins and
deficits in neuropsychological functioning at the
age of 7 to 10 years.
Thompson et al., NEJM, 2007
Vaccines and Autism
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Study of time trends in the prevalence by age
and birth cohort of children with autism (19952007).
The estimated prevalence in children aged 3 to
5 years with autism increased for each quarter
from 1995 through 2007.
No evidence for a recent decrease in autism in
California despite the exclusion of thimerosal
from nearly all childhood vaccines since 2001.
Schechter and Grether, Arch Gen Psych 65:1-24, 2008
Thimerosal
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Thimerosal has been used as a preservative in
many vaccines since the 1930s.
Infants undergoing the usual U.S. program of
vaccines (0 – 6 months) would receive > 0.1 μg
Hg/Kg/day, exceeding the RfD.
The EPA guideline is based on epidemiologic
data on prenatal exposure to MeHg rather than
postnatal exposure to EtHg.
EtHg has some similarities to MeHg.
Structurally related chemicals.
Have a similar initial distribution in the body, and
cause similar types of damage to the brain in
toxic doses.
MeHg vs. EtHg
EtHg decomposes faster than MeHg
Passage through BBB favors small molecules, MeHg
actively transported (EtHg?)
Hg clears faster after administration of EtHg vs. MeHg
Because metabolic rates (basic metabolism, rates of
loss from the body burden) are related to the fractional
power of body weight (allometric relationship), Hg
clears faster from infants
Blood Hg concentrations for MeHg underestimate the
safe exposure range for EtHg.
MeHg is not a suitable reference for risk assessment
from exposure to thimerosal derived Hg.
Comparison of predicted and observed mean
blood total Hg during and after 4 weekly oral
doses (20 µg/kg) of MeHg
Burbacher
et al., EHP
2005
Comparison of predicted and observed mean
blood total Hg concentration during and after
4 weekly i.m. injection of vaccine containing
thimerosal at 20 µg/kg of Hg
Burbacher
et al., EHP
2005
Washout of total Hg in blood and the brain
after 4 weekly oral MeHg doses (20 µg/kg; A)
and 4 weekly im injections of thimerosal (20
µg/kg of Hg; B).
A
B
Burbacher et al., EHP 2005
Washout of organic and inorganic Hg in the
brain after 4 weekly oral MeHg doses (20
µg/kg; A) and 4 weekly im injection of
thimerosal (20 µg/kg of Hg; B).
A
B
Burbacher et al., EHP 2005
Newborn infants
2-month-old infants
6-month-old infants
Blood mercury levels
before and after
receipt of vaccines
that contained
thimerosal
preservative
Blood mercury half-life
approximates 3.7 days
and returns to
prevaccination levels by
day 30.
Pichichero et al., Pediatrics, 2008
Autism and Metals
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Determined the level of mercury, lead, and zinc in
baby teeth of children with ASD
Children with autism had significantly (2.1-fold)
higher levels of mercury but similar levels of lead
and similar levels of zinc.
Children with autism had significantly higher
usage of oral antibiotics during their first 12-36
mo of life.
Antibiotic use is known to almost completely
inhibit excretion of mercury due to alteration of
gut flora.
Adams et al., J Toxicol Environ Health, 2007
Autism and Metals
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Examined the difference between sulfhydrylreactive metals (mercury, lead, arsenic, and
cadmium) in the hair of 45 children with autism
(1-6 yr of age) and matched controls.
Arsenic, cadmium, and lead were significantly
lower in the hair of children with autism than in
matched controls.
Mercury was in the same direction (lower in
autism) following the same pattern, but did not
achieve statistical significance.
Kern et al., J Toxicol Environ Health, 2007
Autism and Metals
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Used data from the California autism surveillance
system to estimate hazardous air pollutant (HAP)
concentrations compiled by the EPA.
284 children with ASD and 657 controls, born in
1994 in the San Francisco Bay area.
Adjusted odds ratios (AORs).
The individual compounds that contributed most
to associations with ASD included mercury,
cadmium, and nickel.
Windham et al., Environ Health Perspect, 2006
Autism and Metals
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Concentration levels of antimony, uranium,
arsenic, beryllium, mercury, cadmium, lead and
aluminum from 40 boys with autism and 40
healthy boys.
The children with autism had significantly
(p<0.001) higher in-hair concentration levels of
lead, mercury and uranium.
There was no significant difference between the
two groups in the other five toxic elements.
Fido and Al-Saad, Autism, 2005
Autism and Mercury Excretion
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Urinary mercury excretion measured following
chelation with succimer (DMSA) was evaluated
in 221 children with ASD and unmatched
controls.
Post-chelation mercury concentrations in the
urine of the autistic cases was approximately 3
times higher.
Bradstreet, 2003
Autism and Hair Mercury Levels
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First baby haircut samples from 94 children with
autism and 45 age- and gender-matched controls.
Within the autistic group, hair mercury levels varied
significantly across mildly, moderately, and severely
autistic children, ( 0.79, 0.46, & 0.21 ppm,
respectively).
Hair Hg levels among controls were significantly
correlated with the number of the mothers' amalgam
fillings and their fish consumption as well as exposure
to mercury through childhood vaccines, correlations
that were absent in the autistic group.
Hair excretion patterns among autistic infants were
significantly reduced relative to control.
Holmes et al., Int J Toxicol 2003
General Considerations
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Urinary mercury excretion in autistic cases could
reflect higher current exposure to mercury rather
than increased tissue retention.
Selection bias
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controls used in these types of studies are not randomly
selected, but rather, chosen from a population of
children whose parents brought them to the clinic for
elective determination of mercury levels.
parental concern about potential mercury toxicity lead
them to restrict the child’s exposure to mercury
(seafood).
General Considerations
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Holmes study, 46% of mothers in the case group
reported being treated with Rho D immunoglobulin during pregnancy; high proportion vs. the
control group and population estimates.
Could indicate substantial recall bias in maternal
reports (which were not validated through review
of medical records) and/or a highly selected and
unrepresentative group of cases.
General Considerations
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No prechelation levels reported; impossible to
tell whether chelation has an effect on Hg
excretion.
No information provided on present or past Hg
exposure in either the cases or the controls.
Concern about lack of standardization in
collection procedures (hair and urine)
Testing not conducted in a blinded fashion
Data unreliable and limited, better designed
studies needed.
Rate of Vaccination or
MMR vaccine
Autistic
vs.
Non-autistic
Madsen et al., NEJM 347, 1477-1482, 2002
Smeeth et al., Lancet 364, 963-969, 2004
Rate of Autism
Vaccinated
Non-vaccinated
Not feasible; Autism prevalence relatively low and
rates of unvaccinated kids are lower still
Hypothesis
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Inherited variations in metal
transporters or metal
clearance genes may render
certain individuals more
susceptible to Hg toxicity and,
in the context of environmental
exposure to Hg, are
associated with autism.
Metal-regulatory genes for Hg
transport and clearance
Metal transporters:
• LAT1: L-type neutral amino acid transporter 1
• DMT1: divalent metal transporter 1
Metal clearance genes:
• MTF1: metal-regulatory transcription factor 1
SNP rs3790625 previously reported in Asian samples
to be associated with ASD (Serajee, et al. 2004)
• MT1a: metallothionein 1a
Methods
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Design primers to PCR amplify:
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Screen for genetic variation
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All exons, exon-intron boundaries, 5′
and 3′ untranslated regions, and up to
1000 bp of the promoter region
Single Strand Conformation
Polymorphism (SSCP) Gel
Electrophoresis and Sequencing
24 cases, 24 controls
Fisher’s exact test
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P<0.05
Summary
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64 polymorphisms currently identified
 27 are novel
 2 synonymous SNPs (same amino acid)
 2 nonsynonymous SNPs (amino acid change)
Polymorphisms in LAT1, DMT1, MTF1 and MT1a
do not exhibit allele frequencies or genotype
frequencies that differ significantly between
autistic and control populations.
In the process in examining 4 variants (1 LAT1; 3
DMT1) in a larger data set (n=220) using
TaqMan assays.
For additional details please see Dr. Sarah Owens’s Poster
Acknowledgements
Sarah E. Owens, Ph.D.
Marshall Summar, M.D.
Jonathan Haines, Ph.D.
Kelli Ryckman
National Institute of Environmental
Health Sciences (NIEHS) 07331
Thompson et al., NEJM, 2007
Thompson et al., NEJM, 2007