Environmental Exposures in Autism Etiology Irva Hertz-Picciotto University of California Davis,...

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

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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

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are needed to see this picture.

QuickTime™ and aTIFF (Uncompressed) decompressor

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QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Pet flea/tick shampoos

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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