Cynthia F' Bearer, M'D', Ph'D'

1
Detecting the Impact of Ethanol on the Fetus and
Understanding its Underlying Mechanism

• Cynthia F. Bearer, M.D., Ph.D.
• Associate Professor
• Departments of Pediatrics, Neurosciences
  Environmental Health Sciences
• Case Western Reserve University

2
Perfect Outcome of Pregnancy
3
Ethanol Is the Leading Known Cause of Mental
Retardation

• Incidence of FAS 1/3,000 live births
• 1 of all live births manifest FAS or some
  prenatal alcohol damage
• FAS and FASD estimated to cost 75 million to
  9.7 billion dollars a year

4
Biomarkers
Biomarker of Effects
Biologically Effective Dose
Early Biological Effect
Altered Structure/ Function
Internal Dose
Out- comes
Exposure
Biomarker of Susceptibility
5
Biomarkers

• Indicators of an event in biologic systems or
  samples
• Three categories
• Exposure (Blood lead level)
• Effect (Liver function tests)
• Susceptibility (Genes)

6
Biomarkers

• Need to identify a biological sample in which to
  measure the biomarker
• Need to identify a biomarker
• Native chemical
• Metabolite or adduct
• Enzyme induction
• New protein expression
• Validation of the biomarker

7
Practical Limitations of Biomarkers in Humans

• Noninvasive
• Readily acceptable to test subjects
• Inexpensive
• Sensitive

8
Potential Biological Samples For Fetal Exposure

• Maternal specimens
• Urine
• Hair
• Blood
• Breath
• Transdermal
• Maternal/fetal/neonatal imaging

9
Potential Biological Samples For Fetal Exposure

• Newborn Specimens
• Cord blood
• Placenta and/or cord
• Urine
• Hair
• Breath
• Transdermal
• Amniotic Fluid
• Meconium

10
Fatty Acid Ethyl Esters(FAEE)

• R-C-OH HO- CH2-CH3 R-C-O-CH2-CH3
• R 120, 140, 160, 181, 182, 183, 204,
  226
• Long half life
• Found in fetal tissues
• ? Role in pathogenesis

11
FAEE Biomarkers of Prenatal Ethanol Exposure
GC/FID
Signal strength
Retention time, min.
12
Question 1 Do women who drink during pregnancy
have more FAEE in their babies meconium?
13
Background

• Cleveland population
• 219 meconium samples with maternal history
• Significant difference between reported drinking
  of mothers who tested positive or negative for
  ethyl linoleate in meconium

Drinks/week, Month prior
P 0.006
Bearer et al, Alcohol Clin Exp Res, 1999
14
Question 2Are FAEE in meconium a clinically
useful test to identify women who drink during
their pregnancy?
15
Amish

• They drink!!!!

16
Muslims

• They dont drink!!!

17
Compare FAEE between groups to identify
informative FAEE

• J all p lt 0.0001 from Jordan samples
• CA all plt0.05 from Cleveland abstainers

Bearer et al, J Pediatr 2005
18
7 Drinks/drinking dayMonth prior to pregnancy
Bearer et al, J Pediatr 2005
19
OUT OF AFRICA

• Capetown Province has the highest known
  incidence of FAS in the world
• 5 - 8 of all liveborns in one area of South
  Africa are affected

20
SOUTH AFRICAN STUDY

• Prospective detailed alcohol use questionnaires
  in a population where denial is not expected to
  be as pronounced
• Degree of drinking much larger than in Cleveland
  sample
• Meconium analysis done by GC/MS/MS at CDC (more
  sensitive/specific)

21
Correlations of Self-Reported Alcohol Consumption
with FAEE
N AADY AADD
DDY/WK Adjusted for wet weight Ethyl palmitate
26 0.25 0.34 0.29 Ethyl oleate
25 0.34 0.48 0.32 Ethyl
linoleate 27 0.26 0.27 0.29 Adjusted
for dry weight Ethyl palmitate 26 0.20
0.35 0.24 Ethyl oleate 25 0.29
0.51 0.24 Ethyl linoleate 27
0.21 0.27 0.24 p lt 0.10, p lt 0.05,
p lt 0.01.
Bearer et al, J Pediatr 2003
22
Correlations Of Self-reported AADD By Trimester
With Concentrations Of Ethyl Oleate In Meconium
N Pre 1st 2nd 3rd Ave. Ethyl oleate
Wet weight 25 .29 .38 .52 .42 .48
Dry weight 25 .32 .42 .55 .40 .51 p lt
0.10, p lt 0.05, p lt 0.01.
Bearer et al, J Pediatr 2003
23
SCATTERGRAM
Bearer et al, J Pediatr 2003
24
ROC Curve 3 drinks/episode
Bearer et al, J Pediatr 2003
25
Ethyl Oleate - South Africa3 drinks/episode
Bearer et al, J Pediatr 2003
26
FAEE in Sheep Meconium
Littner, submitted Pediatr Res
27
Question 3Can FAEE in meconium identify at risk
newborns?
28
ClevelandAssociation of FAEE with MDI and PDI
after controlling for other variables
Peterson, J Pediatr, in revision
29
South Africa
30
South Africa
31
ACKNOWLEDGEMENTS

• Case
• L.T. Singer
• S. Minnes
• H.L. Kirchner
• M.A. ORiordan
• L.M. Santiago
• Y. Littner
• J. Peterson
• Wayne State U.
• S.W. Jacobson
• J.L. Jacobson
• J. Croxford
• CDC
• D.L. Barr
• CHMCC
• B. Lanphear

• U. of Cape Town
• C.D. Molteno
• A. Hay
• A.S. Marais
• University of Witwatersrand
• D.L. Viljoen
• Stanford University
• H.E. Hoyme
• SUNY Buffalo
• L.K. Robinson
• Texas AM University
• T. Cudd
• Harvard University
• R.C. Carter

Funded by The Arc, NIAAA, NIEHS, CDC/AAMC
32
Striking overlap between patients with FAS/ARND
and L1 mutations

• FAS/FASD and L1 mutations
• Agenesis/hypoplasia of the corpus callosum
• Enlarged ventricles, hydrocephalus
• Nystagmus, ptosis, optic n. hypoplasia
• Abnormal migration of corticospinal neurons
  and/or hypoplasia of the corticospinal tract
• Hypoplasia of cerebellar vermis

33
What is L1?

• Transmembrane glycolipoprotein
• Highly conserved member of Ig Superfamily
• Expressed only on postmitotic neurons
• Distributed only on axons and growth cones in the
  developing animal and unmyelinated neurons in the
  adult
• Functions Binding, neurite outgrowth, signal
  transduction
• Charness, Wilkemeyer ethanol inhibits L1-L1
  cell adhesion

34
Ethanol Inhibits L1 Mediated Neurite Outgrowth
Neurite Outgrowth
35
Neurite outgrowth is inhibited by ethanol
PD6 rat Cerebellar Granule Neurons
Bearer et al, JBC, 1999
36
Red L1, Green - NCAM
Control 100 mM Ethanol
Yeaney, et al, JNeurochem in revision
37
GROWTH CONE
AXON
L1-pYRSLE
PERK
2
1
FILOPODIA
ERK
3
L1-YRSLE
p60src
L1-pYRSLE
L1-YRSLE
4/5
38
Our Findings

• Ethanol did not change sorting to the leading
  edge of the growth cone
• Ethanol inhibited L1 mediated ERK activation
• Ethanol inhibited L1 mediated p60src activation
• Ethanol inhibited L1 mediated dephosphorylation
  of L1
• Ethanol inhibited L1 mediated L1 tyrosine
  phosphorylation

39
Clustering of L1 activates ERK which is inhibited
by ethanol
Tang et al, J Neurochem, 2006
40
P0.006
P0.025
Tang et al, J Neurochem, 2006
41
GROWTH CONE
AXON
L1-pYRSLE
PERK
2
1
FILOPODIA
ERK
3
L1-YRSLE
p60src
Lipid Raft
L1-pYRSLE
L1-YRSLE
4/5
42
Lipid rafts

• Specialized microdomains of the plasma membrane
• Serve as platforms for protein-protein
  interaction
• Rich in phosphatidyl-
• choline and GM1 ganglioside
• Disruption reduces neurite outgrowth on L1 and
  N-cadherin but not laminin
• Role in neurotoxicity?

43
Are lipid rafts a site of ethanol toxicity?
44
EtOH does not disperse lipid rafts
1 2 3 4 5 6 7 8
9 10
-TfR
-CTXB
Control
-TfR
-CTXB
EtOH, 25 mM
45
Ethanol did not change N-Cadherin distribution in
lipid rafts
46
Ethanol significantly increases the proportion of
L1 in lipid rafts
47
Confocal image Cells without treatment of 25 mM
Ethanol
L1CD CTxB
Merge
48
Confocal image Cells with treatment of 25 mM
Ethanol
L1CD CTxB
Merge
(Yellow color indicates overlap)
49
Cut-off of effect between butanol and pentanol
L1
N LR N LR N
LR N LR Control Ethanol
Butanol Pentanol
50
Cut-off of effect between butanol and pentanol
0.003
0.02
51
Dose response similar between L1 distribution in
LR and neurite outgrowth
52
HypothesisIf ethanol targets protein-lipid raft
interactions, then abolishing lipid rafts would
abolish ethanol effects
53
Cholesterol depletion abolishes ethanol effect
54
Choline and GM1 ganglioside have been shown to be
neuroprotective

• Choline given either with ethanol or after
  ethanol improved outcomes in exposed rats
• GM1 gangliosides supplemention prevented ethanol
  induced cortical cell plasma membrane alterations
  in vitro

55
Choline and GM1 ganglioside supplementation
prevent ethanol induced redistribution of L1 in
lipid rafts
56
Speculation

• We speculate that an underlying mechanism of
  ethanol toxicity is its effect on lipid
  raft-protein interactions
• A potential therapeutic target is the lipid raft

57
Acknowledgements

• Bearer Lab
• Ningfeng Tang
• Min He
• Natalie Yeaney
• Ben Farah
• Kevin Buck
• Stephanie Fox

• Vance Lemmon
• V. Lemmon Lab
• Mary Ann ORiordan
• Al Malouf

Supported by NIH AA011839