Environmental Exposures in Autism Etiology
Irva Hertz-PicciottoUniversity of California Davis,
M.I.N.D. InstituteCenter 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
A B
CB D E
Most rare: one cause
Most common: 3 or more causes
Possible: 2 causes
F
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
F BE
G
A
C
B
D
HD
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
1. Pesticides
2. Metals
3. Organic pollutants(PCBs, PBDEs, phthalates)
4. Viruses, bacteria & other infections
5. Medical procedures & pharmaceuticals
6. Nutritional factors
Biospecimens: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?DietResidential informationLifestyleConsumer productsMedical history
Linkage to exposure databasesAir, water, pesticides, haz wasteGIS
Medical records: Completeness?
DESIGN & METHODS
Population-based recruitmentClinical confirmation of dx
Linkage to state-of-the-art laboratories
CHARGE Study Design:Case-control - three groups 1. Children with autism
2. Children with developmental delay
3. Children drawn from general population of births, frequency-matched on age, gender & geography
California DDS
California Birth files
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 aTIFF (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
The CHARGE Study
Leptin: a cytokine Figure 1
P<0.0066 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
Gregg et al., Genomics 2008;91:22-29
Early Onset Autism (A-E) vs General Population (GP)
Differential gene expression in autism compared to typically developing,
general population controlsFold-change
Gene Affymetrix GeneChip Taqman p-value
PAM 1.86 1.51 0.007SPON2 1.87 1.86 0.005IL2RB 1.56 1.35 0.046PRF1 1.79 1.53 0.027GZMB 2.01 1.72 0.014CX3CR1 1.60 1.37 0.006SH2D1B/EAT2 2.19 1.78 0.011EDG8 1.99 ND –IGFBP7 1.70 ND –ZNF145 1.99 ND –KIR3DL2 1.58 ND
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
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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
• 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.
Dysmorphology
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
Wiest et al., in submission
Docosahexanoic acid (DHA) • abundant in phospholipids of brain and retina• contributes to
membrane structure and functioneicosanoid signalinggene expression modulation
Other Functions:• Inhibition of neuronal apoptosis and• Regulating neuronal excitability through GABA receptors
Developing brains obtain DHA from circulation
RESULTS
Environmental Chemicals
• 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
Hg and Autism
The CHARGE StudyMercury Levels by Diagnostic Group (N=556)
0
5
10
15
20
25
30
35
< -4.00 -4.00 to -3.01
-3.00 to -2.01
-2.00 to -1.01
-1.00 to -0.01
0.00 to0.99
1.00 to1.99
≥ 2.00
log Hg (ug/L)
% Children
ASD(n=327)
Delayed(n=65)
Typical(n=164)
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
PBDEsPolybrominated 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
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
Pet flea/tick shampoos
QuickTime™ and aTIFF (Uncompressed) decompressorare needed to see this picture.
EctoKyl CA Pyrethrin Flea Shampoo - 12 oz.
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.
Hartz 2 in 1 Rid Flea Dog Shampoo with Pyrethrin - 18
oz.
Bio-Groom Flea & Tick
Pyrethrin Spray16 fl 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
LMP1st 2nd 3rd
Delivery 1 year
Brain Development
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 self-report: 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
LMP1st 2nd 3rd
Delivery 1 year
Brain Development
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 PessahRobin HansenCheryl WalkerJudy Van de WaterPaul AshwoodJeffrey GreggFrank SharpBruce GermanBruce HammockPeter GreenRobert BermanDanh Nguyen
• UC DavisFlora TassoneLihong QiJanine LasalleSally Ozonoff
• Univ MinnessotaScott Selleck
• VanderbiltPat Levitt
• USC (Southern Calif)Rob McConnell
• Johns HopkinsLi-Ching Lee
Be in CHARGE !
http://beincharge.ucdavis.edu/
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
Grateful thanks to:
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