Michael Aschner

Dept of Pediatrics & Pharmacology, & the Kennedy Center for Research on Human

Development, Vanderbilt University Medical Center, Nashville, TN

Metals in ASDMetals in ASD

OutlineOutline

VaccinesVaccinesMethylmercury vs ethylmercury (thimerosal)Methylmercury vs ethylmercury (thimerosal)

Other metals in ASD Other metals in ASD General considerations on the validity of the General considerations on the validity of the

studies – research needsstudies – research needsPolymorphisms in “metal handling” genes Polymorphisms in “metal handling” genes

and ASDand ASD

Thimerosal-Containing Vaccines and Thimerosal-Containing Vaccines and Autistic Spectrum DisorderAutistic Spectrum Disorder

Parker et al., Pediatrics 114:793-804, 2004

Vaccines and AutismVaccines and Autism

Two studies performed in the UK examined whether thimerosal in vaccines caused neurodevelopmental or psychological problems (Parker et al., Pediatrics, 2004); 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 Mercury and neuropsychological outcomesoutcomes

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

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 Higher prenatal Hg exposure was associated with better performance on one measure of language better performance on one measure of language and poorer performance on one measure of and poorer performance on one measure of attention and executive functioning.attention and executive functioning.

Increasing levels of Hg exposure from birth to 7 Increasing levels of Hg exposure from birth to 7 months were associated with better performance on months were associated with better performance on one measure of fine motor coordination and on one one measure of fine motor coordination and on one measure of attention and executive functioning.measure of attention and executive functioning.

Mercury and neuropsychological Mercury and neuropsychological outcomesoutcomes

Thompson et al., NEJM, 2007

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 thimerosal-containing vaccines and immune globulins and deficits in neuropsychological functioning at the age of 7 to 10 years.

Mercury and neuropsychological Mercury and neuropsychological outcomesoutcomes

Thompson et al., NEJM, 2007

Study of time trends in the prevalence by age and birth cohort of children with autism (1995- 2007).

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.

Vaccines and AutismVaccines and Autism

Schechter and Grether, Arch Gen Psych 65:1-24, 2008

ThimerosalThimerosal 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. EtHgMeHg vs. EtHgEtHg decomposes faster than MeHgPassage through BBB favors small molecules, MeHg actively transported (EtHg?)Hg clears faster after administration of EtHg vs. MeHgBecause 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 infantsBlood 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 Comparison of predicted and observed mean blood total Hg during and after 4 weekly oral blood total Hg during and after 4 weekly oral

doses (20 µg/kg) of MeHgdoses (20 µg/kg) of MeHg

Burbacher et al., EHP 2005

Comparison of predicted and observed mean Comparison of predicted and observed mean blood total Hg concentration during and after blood total Hg concentration during and after 4 weekly i.m. injection of vaccine containing 4 weekly i.m. injection of vaccine containing

thimerosal at 20 µg/kg of Hgthimerosal at 20 µg/kg of Hg

Burbacher et al., EHP 2005

Washout of total Hg in blood and the brain Washout of total Hg in blood and the brain after 4 weekly oral MeHg doses (20 µg/kg; A) after 4 weekly oral MeHg doses (20 µg/kg; A) and 4 weekly im injections of thimerosal (20 and 4 weekly im injections of thimerosal (20

µg/kg of Hg; B). µg/kg of Hg; B).

Burbacher et al., EHP 2005

A B

Washout of organic and inorganic Hg in the Washout of organic and inorganic Hg in the brain after 4 weekly oral MeHg doses (20 brain after 4 weekly oral MeHg doses (20

µg/kg; A) and 4 weekly im injection of µg/kg; A) and 4 weekly im injection of thimerosal (20 µg/kg of Hg; B). thimerosal (20 µg/kg of Hg; B).

Burbacher et al., EHP 2005

A B

Blood mercury levels Blood mercury levels before and after before and after

receipt of vaccines receipt of vaccines that contained that contained

thimerosal thimerosal preservative preservative

Pichichero et al., Pediatrics, 2008

Blood mercury half-life Blood mercury half-life approximates 3.7 days approximates 3.7 days and returns to and returns to prevaccination levels by prevaccination levels by day 30. day 30.

Newborn infantsNewborn infants

2-month-old infants2-month-old infants

6-month-old infants6-month-old infants

Autism and MetalsAutism and Metals 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 MetalsAutism and Metals Examined the difference between sulfhydryl-Examined the difference between sulfhydryl-

reactive metals (mercury, lead, arsenic, and reactive metals (mercury, lead, arsenic, and cadmium) in the hair of 45 children with autism cadmium) in the hair of 45 children with autism (1-6 yr of age) and matched controls. (1-6 yr of age) and matched controls.

Arsenic, cadmium, and lead were significantly Arsenic, cadmium, and lead were significantly lower in the hair of children with autism than in lower in the hair of children with autism than in matched controls.matched controls.

Mercury was in the same direction (lower in Mercury was in the same direction (lower in autism) following the same pattern, but did not autism) following the same pattern, but did not achieve statistical significance. achieve statistical significance.

Kern et al., J Toxicol Environ Health, 2007

Autism and MetalsAutism and Metals

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 MetalsAutism and Metals

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 ExcretionAutism and Mercury Excretion 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 LevelsAutism and Hair Mercury Levels

Holmes et al., Int J Toxicol 2003

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.

General ConsiderationsGeneral Considerations Urinary mercury excretion in autistic cases could

reflect higher current exposure to mercury rather than increased tissue retention.

Selection bias 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).

Holmes study, 46% of mothers in the case group reported being treated with Rho D immunoglo-bulin 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 ConsiderationsGeneral Considerations

General ConsiderationsGeneral Considerations

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 Rate of Vaccination or MMR vaccineMMR vaccine

Rate of AutismRate of Autism

Autistic vs. Non-autistic

Vaccinated Non-vaccinated

Madsen et al., NEJM 347, 1477-1482, 2002

Smeeth et al., Lancet 364, 963-969, 2004

Not feasible; Autism prevalence relatively low and rates of unvaccinated kids are lower still

HypothesisHypothesis

Inherited variations in metal Inherited variations in metal transporters or metal transporters or metal clearance genes may render clearance genes may render certain individuals more certain individuals more susceptible to Hg toxicity and, susceptible to Hg toxicity and, in the context of environmental in the context of environmental exposure to Hg, are exposure to Hg, are associated with autism.associated with autism.

Metal-regulatory genes for Hg Metal-regulatory genes for Hg transport and clearancetransport 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

MethodsMethods

Design primers to PCR amplify:Design primers to PCR amplify: All exons, exon-intron boundaries, 5′ and

3′ untranslated regions, and up to 1000 bp of the promoter region

Screen for genetic variationScreen for genetic variation Single Strand Conformation Single Strand Conformation

Polymorphism (SSCP) Gel Polymorphism (SSCP) Gel Electrophoresis and SequencingElectrophoresis and Sequencing

24 cases, 24 controls24 cases, 24 controls Fisher’s exact test

P<0.05P<0.05

SummarySummary 64 polymorphisms currently identified64 polymorphisms currently identified

27 are novel2 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 In the process in examining 4 variants (1 LAT1LAT1; 3 ; 3 DMT1DMT1) in a larger data set (n=220) using ) in a larger data set (n=220) using TaqMan assays.TaqMan assays.

For additional details please see Dr. Sarah Owens’s Poster

AcknowledgementsAcknowledgements

Sarah E. Owens, Ph.D.

Marshall Summar, M.D.Marshall Summar, M.D.

Jonathan Haines, Ph.D.Jonathan Haines, Ph.D.

Kelli Ryckman

National Institute of Environmental National Institute of Environmental Health Sciences (NIEHS) 07331Health Sciences (NIEHS) 07331

Thompson et al., NEJM, 2007

Thompson et al., NEJM, 2007