Environmental Exposures
in Autism Etiology
Irva Hertz-Picciotto
University of California Davis,
M.I.N.D. Institute
Center for Children’s Environmental Health
& Department of Public Health Sciences
Mechanisms for Environmental
Chemicals to Affect Autism Risk
• Direct action on neural tissue during:
–
–
–
–
Differentiation
Migration
Dendritic branching/pruning
Synaptogenesis
• Effects on genes that regulate CNS development
• Perturbation of immune signaling/inflammation
via neurotransmitters &/or cytokines
• Endocrine disruption: sex steroids/thyroid
hormones
Causes Unknown but Numerous
Autism is multifactorial, meaning:
multiple causes across the population and
within an individual
B
A
D
C
Possible: 2 causes
B
F
Most common: 3
or more causes
E
Most rare:
one cause
Genes & Environment in Autism?
• Genetics: ~60-90% (twin studies)
• Environment: 10-40% = minimum
• Sum not 100%
• Risk Ratios of 10+:
– Congenital rubella
– Thalidomide
• Risk Ratios ~4 to 5:
– Maternal age
– Male sex
A
B
C
D
E B
F
G
D
H
Center for Children’s
Environmental Health (CCEH)
• NIEHS call in 2000
• State of the epidemiologic literature on risk factors
–
–
–
–
Descriptive male:female
Obstetric suboptimality
Twin studies & familial aggregation
Reported trend towards increasing prevalence
• Environmental causes: viral, pharmacologic
• Need for multi-disciplinary approach
• UC Davis CCEH: The CHARGE Study
The CHARGE* Study
*CHildhood Autism Risk from
Genetics and the Environment
Goals of the CHARGE Study
To identify causes and contributing risk &
protective factors for childhood autism:
• Genetic susceptibility
• Environmental exposures
• Interactions of the two
To determine mechanisms of susceptibility and
identify subsets of possible distinct etiologies for
childhood autism
• Immunologic
• Genetic/Genomic
• Metabolic/metabolomic
• Phenotypic
Environmental Exposures
Biospecimens:
1. Pesticides
2. Metals
3. Organic pollutants
(PCBs, PBDEs,
phthalates)
4. Viruses, bacteria &
other infections
5. Medical procedures
& pharmaceuticals
6. Nutritional factors
Blood (recent exposures)
Child’s hair (recent - depends on length)
Baby lock (first year of life)
Mother’s hair (if long enough, prenatal)
Urine (recent exposure, metabolism)
Newborn blood spot (prenatal)
Interviews: Recall?
Diet
Residential information
Lifestyle
Consumer products
Medical history
Linkage to exposure databases
Air, water, pesticides, haz waste
GIS
Medical records: Completeness?
DESIGN & METHODS
Population-based recruitment
Clinical confirmation of dx
Linkage to state-of-the-art laboratories
CHARGE Study Design:
Case-control - three groups
California
DDS
California
Birth files
1.
2.
3.
Children with autism
Children with developmental delay
Children drawn from general population of
births, frequency-matched on age, gender &
geography
All children - Eligibility: 24-60 months, born in
Calif, mother English/Spanish speaking, living
with biologic parents
The CHARGE Study Clinical Protocol
Confirmation of diagnosis:
• ADI and ADOS (autism cases only)
• Social Communication Questionnaire (all others)
Cognitive & adaptive development (all children)
Medical Exam
Medical Hx
Parent forms
Medical records obtained
Environmental Exposure Questionnaire
The CHARGE Study Clinical Protocol
• Specimens:
- blood
- urine
- hair
…from index child, parents & siblings
• Newborn blood spots from CDPH GDB
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
RESULTS
Immunologic
Deviations
Maternal Antibodies to
Fetal Brain Tissue (n=163)
Braunschweig et al., Neurotoxicology 2008; 29:226-23
Leptin: a cytokine
Figure 1
The CHARGE
Study P<0.006
6
P<0.012
P<0.026
N=70
N=50
N=23
N=26
Ashwood et al., J Autism Dev Disorders 2008;38:169-175
RESULTS
Genomics, Genetics &
Epigenetics
Early Onset Autism (A-E) vs General Population (GP)
Gregg et al., Genomics 2008;91:22-29
Differential gene expression in autism
compared to typically developing,
general population controls
Gene
Fold-change
Affymetrix GeneChip Taqman
PAM
SPON2
IL2RB
PRF1
GZMB
CX3CR1
SH2D1B/EAT 2
EDG8
IGFBP7
ZNF145
KIR3DL2
1.86
1.87
1.56
1.79
2.01
1.60
2.19
1.99
1.70
1.99
1.58
1.51
1.86
1.35
1.53
1.72
1.37
1.78
ND
ND
ND
ND
p-value
0.007
0.005
0.046
0.027
0.014
0.006
0.011
–
–
–
Early Onset Autism vs GP:
Cell Types
227018_at (MSTP141; FLJ14920; FL...
1554638_at (ENDOFIN; KIAA0305; D...
235507_at (FLJ10883)
224429_x_at (MGC10997)
241354_at (FLJ13330)
201883_s_at (GT1; GTB; GGTB2; B4...
233713_at (HSKM-B; ZMYND14; MG...
209128_s_at (TIP110; KIAA0156; p11...
212904_at (KIAA1185; RP1-286D6.3)
201697_s_at (DNMT; MCMT; CXXC9; ...
210164_at (HLP; CCPI; CGL1; CSPB;...
202786_at (DCHT; SPAK)
201080_at (Pip4k2B; PIP5KIIB; PIP5...
230464_at (S1P5; Edg-8; S1PR5; SP...
205291_at (P70-75)
1553681_a_at (P1; PFP; HPLH2; MG...
206118_at (STAT4)
228854_at
1553177_at (EAT2)
233558_s_at (FLJ12716)
217776_at (MDT1; PSDR1; RALR1; S...
209123_at (DHPR; PKU2)
202783_at (MGC126502; MGC12650...
217301_x_at (RBAP48)
200698_at (ELP-1; ERD2.2)
221517_s_at (MED17; CRSP77; DRI...
215743_at (NMT2)
219178_at (FLJ12960)
217761_at (MTCBP-1; SIPL; FLJ1091...
217878_s_at (APC3; HNUC; D0S143...
216153_x_at (ST15; hRECK)
PMN Mono B
CD4 CD8
NK
Gregg et al., Genomics 2008;91:22-29
Other Related Studies
• Candidate genes (Tassone)
MAOA, 5HT-receptor, GSTM1
• SNPs (Gregg)
• Copy Number Variants (Selleck)
• Epigenetics (Lasalle) - X-Chromosome
Inactivation
Dysmorphology
•
•
•
•
•
•
Digital photos are taken of face: frontal and profile views
Blind review by experts in genetics & pediatrics
Identify minor physical anomalies
Disagreements resolved by consensus of all 3 raters.
242 children were reviewed (121 ASD, 51 DD, and 70 TD)
ASD children more likely to be classified as dysmorphic or
partially dysmorphic.
• Those with 6 or more dysmorphic features may be a
subgroup with a high genetic susceptibility.
Angkustsiri et al., in preparation; presented at Ped Acad Soc 2008
RESULTS
Lipids
Autism and Fatty Acid Profiles (n=250)
Wiest et al., in submission
Autism and Fatty Acid Profiles
Docosahexanoic acid (DHA)
• abundant in phospholipids of brain and retina
• contributes to
membrane structure and function
eicosanoid signaling
gene expression modulation
Other Functions:
• Inhibition of neuronal apoptosis and
• Regulating neuronal excitability through GABA receptors
Developing brains obtain DHA from circulation
Wiest et al., in submission
RESULTS
Environmental
Chemicals
Hg and Autism
• Great interest: known neurotoxin
• Most research focused on thimerosal in vaccines
• Multiple sources:
• Fish
• Dental amalgams
• Broken thermometers, light bulbs
• Nasal sprays, contact lens solution
CHARGE Study approach
• Current blood
• Baby locks (first haircuts)
• Newborn blood spots
• Questionnaire about use of products
The CHARGE Study
Mercury Levels by Diagnostic Group (N=556)
% Children
35
30
ASD
(n=327)
25
Delayed
(n=65)
20
Typical
(n=164)
15
10
5
0
< -4.00
-4.00 to - -3.00 to - -2.00 to - -1.00 to 3.01
2.01
1.01
0.01
log Hg (ug/L)
0.00 to
0.99
1.00 to
1.99
і 2.00
Prediction of log(Blood Hg)*
Beta
P-value
Autism or ASD
-0.01
0.95
Ate tuna (1+ servings/wk)
0.56
0.001
Ate ocean fish ( “ )
0.68
<0.0001
Ate freshwater fish ( “ )
0.57
0.01
Hg amalgams x chew/grind teeth
0.15
0.004
Nasal spray or ear wax removal
0.52
0.02
* Multivariate analysis to adjust for confounding
Subjects weighted to adjust for differential participation by SES
Interpretation/Conclusion
• No difference in current blood levels of Hg
• Does not confirm previous report of higher blood Hg in
children with autism
• Does not address early life exposures as potential causal
factors
• Possible sequestering of ethyl as opposed to methyl Hg
not addressed in this study
• Only 5% of Hg body burden is in circulation
PBDEs
Polybrominated diphenyl ethers
• Flame retardants
• Sweden (1998) and then EU has banned penta and octa-PBDEs
• Rodents: lack of ability to habituate to a novel situation.
All US women's milk samples were contaminated
with PBDEs from 6 to 419 ng/g, lipid, orders of
magnitude higher than levels reported in
European studies, and are the highest reported
worldwide.
Mol Nutr Food Res 2008
Pilot Study Results:
Autism & PBDEs
• Children with ASD do not have higher levels of PBDEs
• Contrary to our hypothesis, their levels of PBDEs are
lower than in typically developing children
• Possibility of reverse causation: behaviors changed?
• High SES: more air flow in home?
• Association with head growth?
• Used a post-dx measurement -- need to measure
PBDE exposures during critical windows of
development
Household items that may
contain deca-PBDEs
Televisions･Cell phones･Fax machines･Audio and
video equipment･Computers･Printers･Scanners･
Photocopiers･Remote controls･Lamp sockets･
Hairdryers･Fans･Upholstered sofas･Upholstered
chairs･Polyurethane foam･Mattresses･Curtains･
Drapes･Carpet padding･Ovens and stoves･Stove
hoods･Refrigerators･Dishwashers･Washing
machines･Clothes dryer･Microwaves･Toasters･
Coffee makers･Water heaters･Wires and cables･
Circuit breakers･Electrical outlets
Pesticides & Autism
• Roberts et al EHP 2007
 Organochlorines applied commercially within 1/2 mile of
mother’s residence at delivery associated with greater risk of
autism.
 Strongest association in weeks 1-8 of gestation
• Eskenazi et al, Basic & Clin Pharm Toxicol 2008
 Organophosphate metabolites (DAPs) in urine collected
during pregnancy and 1st year of life associated with autism
spectrum disorder symptoms.
• These compounds cross the placenta and bioaccumulate.
Pesticides & Autism n=531
• CHARGE Study: self-reported use of household products
(about 2 dozen)
 Use of pet flea or tick soaps/shampoos by mother doubled the
risk that the child developed autism.
 Associations from 3 months prior to conception through first
year of child’s life
• These shampoos are formulated primarily with pyrethrins
Pet flea/tick shampoos
Mode of Action: Pyrethrins affect the nervous
system of insects and result in repeated and
extended firings of the nerves. They do this by
affecting the flow of sodium out of nerve cells
through voltage-sensitive sodium channels.
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
QuickTi me™ and a
TIFF ( Uncompressed) decompressor
are needed to see thi s pi ctur e.
EctoKyl CA Pyrethrin
Flea Shampoo - 12 oz.
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Bio-Groom Flea & Tick
Pyrethrin Spray
16 fl oz.
Hartz 2 in 1 Rid Flea Dog
Shampoo with Pyrethrin - 18 oz.
Toxicities of pyrethroids
• Neuronal cell death in hippocampus
• Mechanisms may involve not only sodium
but also calcium channels
• Wide variation in age sensitivity
• Piperonyl butoxide enhances toxicity
Early life exposure to pyrethrins has
been shown to compromise the
blood-brain barrier in rodents
Labor Induction &/or Augmentation
• Drugs increasingly used to alter the course of labor
• Oxytocin receptors throughout the brain
• Prostaglandins are potent pro-inflammatory
compounds.
Walker et al., in preparation (to be presented at IMFAR 2008)
Critical Window(s) for
Susceptibility?
Conception
LMP
1st
Delivery
2nd
3rd
Brain Development
1 year
Limitations of CHARGE
• Numbers large for this type of study (n>1000), but small
for studying rare exposures, or rare polymorphisms.
• Data on non-medical exposures by retrospective selfreport: possible reporting bias
• Unclear whether differences (e.g., immune) are:
- part of causal pathway
- a result of neuropathology
- a concommitant parallel effect
• Limited specimens that predate the diagnosis
=> Newest epidemiologic study from the UC Davis
Center for Children’s Environmental Health:
MARBLES
Markers of Autism Risk in
Babies—Learning Early Signs
A study of pregnant moms, who already have a child with
autism, following their pregnancies and new child
Eligibility Criteria for
MARBLES
• Be the biological mother or father of a child
diagnosed with autism
• Be pregnant or planning a pregnancy in the
near future
• Live within 2-hours drive of the
Sacramento/Davis area
• Be over the age of 18
Critical Window(s) for
Susceptibility?
Conception
LMP
1st
Delivery
2nd
3rd
Brain Development
1 year
MARBLES Participants…
• Answer questionnaires and telephone interviews about
family medical history, illnesses and medications,
lifestyle, diet, home product use, and other factors that
may influence their baby’s development
• Complete weekly symptom diaries during pregnancy and
monthly diaries during the first year of child’s life
• Donate biological specimens
• Permit collection of environmental samples
Investigators
• UC Davis CCEH
Isaac Pessah
Robin Hansen
Cheryl Walker
Judy Van de Water
Paul Ashwood
Jeffrey Gregg
Frank Sharp
Bruce German
Bruce Hammock
Peter Green
Robert Berman
Danh Nguyen
• UC Davis
Flora Tassone
Lihong Qi
Janine Lasalle
Sally Ozonoff
• Univ Minnessota
Scott Selleck
• Vanderbilt
Pat Levitt
• USC (Southern Calif)
Rob McConnell
• Johns Hopkins
Li-Ching Lee
Be in CHARGE !
http://beincharge.ucdavis.edu/
Grateful thanks to:
Paula Krakowiak, Lora Delwiche,
Lesley Deprey, Carrie Jones,
Beth Goodlin-Jones, Susan Bacalman
Nicole Tartaglia, Steve Nowicki, Jean Sakamura,
Kathleen Angkuststiri
Melissa Rose
Amy Harley, Angelica Guzon,
Eva Long, Danielle Greenfield,
Carola Gutierrez de King,
Susana Gonzales, Caroline Grantz,
Cynthia Contreras, Devon Baird, Matthew Parro,
Alice Baker
Special Thanks to our Funders
•
•
•
•
NIEHS P01 ES11269
NIEHS 1R01 ES015359
NIEHS 1R01 ES 015171
Environmental Protection Agency (EPA), Science
to Achieve Results (STAR) program, R829388
• The M.I.N.D. Institute/UC Davis Matching Funds
• Autism Speaks
• Cure Autism Now