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New Emerging Team on FASD:
Oxidative Stress, Biomarkers & Antioxidant Therapy
Leader: James F. Brien, Queen’s University
Canadian Institutes of Health Research
CIHR NEW EMERGING TEAM ON FASD
Antioxidant Therapy
C
A
Oxidative Stress
B
Biomarkers
Members of NET on FASD
Alan D. Bocking, obstetrics and maternal-fetal physiology,
University of Toronto;
James F. Brien, basic developmental pharmacology &
toxicology, Queen’s University;
Gideon Koren, pediatrics and clinical pharmacology &
toxicology, Hospital for Sick Children, Toronto;
Stephen G. Matthews, developmental neuro-endocrinology,
University of Toronto;
James N. Reynolds, developmental neuroscience,
Queen’s University;
Joanne Rovet, developmental neuropsychology,
Hospital for Sick Children;
Wendy J. Ungar, health economics and population health,
Hospital for Sick Children.
RESEARCH OBJECTIVES
A. To test the hypothesis that oxidative stress is an
important mechanism of the brain injury of FASD;
B. To identify and validate reliable biomarkers for
fetal ethanol exposure at critical periods of
vulnerability during gestation and for the
magnitude of fetal ethanol exposure;
C. To discover and develop innovative antioxidant
treatment strategies for preventing or attenuating
ethanol-induced oxidative stress in fetal life and
decreasing its impact on brain function in
postnatal life.
Coronal Section of the Brain
RESEARCH OBJECTIVES
A. To test the hypothesis that oxidative stress is an
important mechanism of the brain injury of FASD;
B. To identify and validate reliable biomarkers for
fetal ethanol exposure at critical periods of
vulnerability during gestation and for the
magnitude of fetal ethanol exposure;
C. To discover and develop innovative antioxidant
treatment strategies for preventing or attenuating
ethanol-induced oxidative stress in fetal life and
decreasing its impact on brain function in
postnatal life.
OBJECTIVE A
To determine whether oxidative stress is a
mechanism of the brain injury of FASD.
Definition of Oxidative Stress
Oxygen radicals: highly reactive molecules
generated during cell metabolism.
Cell
degradation
Cell
of O2
production
radicals
of O2
radicals
Overabundance of O2 radicals/Oxidative Stress
Proposed Mechanism of Brain Injury of
FASD
Maternal Ingestion of Ethanol
Fetal Brain Exposure to Ethanol
Oxidative Stress / Increased Reactive Oxygen Species

–
H2O2 O2 OH
Damage to Key Cell Molecules
(DNA, Proteins, Membrane Phospholipids)
Neuronal Cell Death
Brain Injury of FASD
Measures of Oxidative Stress
1. Glutathione (GSH):
• Intracellular GSH localized primarily in mitochondria
and cytoplasm.
2. F2-Isoprostanes:
• Prostaglandin F2-like compounds.
• Formed in vivo by nonenzymatic free radicalinduced peroxidation of arachidonic acid.
• Specific and stable products of lipid peroxidation.
8-iso-Prostaglandin F2
HIPPOCAMPUS
Control Offspring
Ethanol Offspring
Experimental Animal Study Design
Timed Pregnant Guinea Pigs
Ethanol
Isocaloric-Sucrose/
(4g/kg MBW/day)
Pair-Feeding
Water
Term Fetus (GD 65)
Hippocampus
[GSH] in mitochondria and cytoplasm
[8-iso-PGF2] in homogenate
Fetal Hippocampus
GLUTATHIONE
[GSH] (mmol/mg protein)
[GSH] (mmol/mg protein)
MITOCHONDRIA
b
5.0
a
a
2.5
0.0
Ethanol Sucrose
Water
TREATMENT
CYTOSOL
60
50
40
30
20
10
0
Ethanol Sucrose Water
TREATMENT
Fetal Hippocampus
8-iso-PGF2
[8-iso-PGF 2]
(pg/mg protein)
250
GD 65
PD 0
200
150
100
50
0
Ethanol
Sucrose
TREATMENT
Water
G. Weaver, University of Colorado at Denver
Apoptosis (Programmed Cell Death)
and Caspase-3
Modified from J.E. Dawson and L.M Winn
CYTOCHROME C (FETAL HIPPOCAMPUS)
Ethanol
Sucrose
ACTIVATED CASPASE-3
IMMUNOREACTIVITY
(% of all Cells)
ACTIVATED CASPASE-3 (FETAL HIPPOCAMPUS)
30
*
20
10
0
Ethanol
Sucrose
Water
TREATMENT
ETHANOL
SUCROSE
WATER
SUMMARY
Chronic ethanol exposure produces in the fetal
hippocampus:
•
•
•
•
depletion of mitochondrial [GSH];
mitochondrial cytochrome c leakage into cytoplasm;
increase in caspase-3 enzymatic activity;
no change in [8-iso-PGF2].
CONCLUSION
Disruption of the mitochondria and consequent
apoptosis play key roles in the mechanism of the
brain injury of FASD involving the hippocampus.
RESEARCH OBJECTIVES
A. To test the hypothesis that oxidative stress is an
important mechanism of the brain injury of FASD;
B. To identify and validate reliable biomarkers for
fetal ethanol exposure at critical periods of
vulnerability during gestation and for the
magnitude of fetal ethanol exposure;
C. To discover and develop innovative antioxidant
treatment strategies for preventing or attenuating
ethanol-induced oxidative stress in fetal life and
decreasing its impact on brain function in
postnatal life.
Individual [FAEE] in meconium of term fetal offspring
of the ethanol and isocaloric-sucrose/pair-fed groups
2.0
Ethanol
Sucrose
1.0
ni
c
A
ra
ch
i
do
c
le
i
O
ea
St
le
ito
m
ric
ic
c
iti
m
Pa
l
Pa
l
ur
ic
0
La
MECONIUM INDIVIDUAL [FAEE]
(nmol/g)
3.0
INDIVIDUAL FAEE
Total [FAEEs] in meconium of term fetal offspring of the
ethanol, isocaloric-sucrose/pair-fed and water groups
MECONIUM TOTAL [FAEEs]
(nmol/g)
4.0
*
3.0
2.0
1.0
0
Ethanol
Sucrose
TREATMENT
Water
RESEARCH OBJECTIVES
A. To test the hypothesis that oxidative stress is an
important mechanism of the brain injury of FASD;
B. To identify and validate reliable biomarkers for
fetal ethanol exposure at critical periods of
vulnerability during gestation and for the
magnitude of fetal ethanol exposure;
C. To discover and develop innovative antioxidant
treatment strategies for preventing or attenuating
ethanol-induced oxidative stress in fetal life and
decreasing its impact on brain function in
postnatal life.
Rationale for Vitamin C + Vitamin E Study
In women at increased risk of pre-eclampsia,
pharmacological doses of vitamin C (1000
mg/day) and vitamin E (400 IU natural-source/day)
starting at 16-22 weeks’ gestation and continued
throughout the second half of pregnancy:
• decreased the occurrence of pre-eclampsia.
• were apparently safe with no obvious adverse
fetal effects.
L.C. Chappell et al., Lancet (1999).
Ethanol
Ethanol
Vitamins C
C+
Vitamins
+ EE
Vehicle
Vehicle
Nutritional Control
Nutritional
Control
VitaminsC
C+
+E
E
Vitamins
Vehicle
Vehicle
Chronic Treatment Regimen:
Daily oral administration of vitamins C (250mg) + E
(100mg) OR vehicle (milk/cream).
Two hours later,
Oral administration of 4g ethanol/kg maternal body
weight OR isocaloric-sucrose/pair-feeding for five
consecutive days, followed by no treatment for two
days, each week.
PD 45: Morris water-maze task for spatial learning and
memory.
MORRIS WATER MAZE
PD0: Brain and hippocampal weights
Ethanol decreased brain weight compared with control; Vitamin C
plus E treatment protected hippocampal weight in ethanol offspring.
Brain Weight (g)
b
2.0
a
b
a
1.5
1.0
0.5
0.0
0.125
Hippocampus Weight/
Brain Weight Ratio
2.5
0.100
a
b
b
E + Veh
S + Vit
b
0.075
0.050
0.025
0.000
E +Vit
E + Veh
S + Vit
Treatment
S + Veh
E + Vit
S + Veh
Treatment
E = Ethanol; S = Sucrose Control; Vit = Vitamins C + E; Veh = Vehicle
PD 45: Morris water maze
Vitamins C + E protected against the ethanol-induced deficit in
retention of new memory (Old Locations).
"New Locations" of Platform
30
a
a
a
b
20
10
0
E + Vit
E + Veh
S + Vit
TREATMENT
S + Veh
AVERAGE TIME REQUIRED TO
LOCATE HIDDEN PLATFORM (S)
AVERAGE TIME REQUIRED TO
LOCATE HIDDEN PLATFORM (s)
Vitamins C + E produced deficits in both acquisition (New Locations)
and retention (Old Locations) of new memory in control offspring.
"Old Locations" of Platform
25
b,c
20
a
a,c
15
d
10
5
0
E + Vit
E + Veh
S + Vit
S + Veh
TREATMENT
E = Ethanol; S = Sucrose Control; Vit = Vitamins C + E; Veh = Vehicle
SUMMARY
Chronic maternal ethanol administration:
•
•
decreased brain weight in the neonate.
impaired offspring performance in the Morris water-maze
task, resulting in deficits in the acquisition and retention of
new memory.
Maternal administration of vitamins C + E:
•
•
•
protected hippocampal weight in ethanol-exposed offspring
at birth.
protected ethanol-exposed offspring from deficit in
retention of new memory.
produced deficits in acquisition and retention of new
memory in control offspring.
CONCLUSION
Vitamins C + E dose studies are being conducted
to determine optimal antioxidant vitamin therapy
for the brain injury of FASD.
CIHR NEW EMERGING TEAM ON FASD
Antioxidant Therapy
C
D
Training
PDF
PhD
MSc
A
B
Oxidative Stress
Biomarkers
Ethanol-induced Oxidative Stress Mechanism
R. Cohen-Kerem & G. Koren, Neurotoxicol. Teratol. (2003).
HIPPOCAMPAL CA1 PYRAMIDAL CELL LOSS
GD 62: No cell loss
PD 1: 25% Cell Loss
PD 5: 30% Cell Loss
PD 12: 30% Cell Loss
McGoey et al., 2003
CLEAVED PARP
IMMUNOREACTIVITY
(% of all Cells)
CLEAVED PARP (FETAL HIPPOCAMPUS)
30
*
20
10
0
Ethanol
Sucrose
Water
TREATMENT
ETHANOL
SUCROSE
WATER
PD 45: Morris water maze
Ethanol and/or vitamins C + E treatment did not affect swim
speed in locating the hidden platform.
250
200
150
100
50
0
E + Vit
E + Veh
S + Vit
TREATMENT
S + Veh
"Old Locations" of Platform
SWIM SPEED IN LOCATING
HIDDEN PLATFORM (cm/s)
SWIM SPEED IN LOCATING
HIDDEN PLATFORM (cm/s)
"New Locations" of Platform
250
200
150
100
50
0
E + Vit
E + Veh
S + Vit
S + Veh
TREATMENT
E = Ethanol; S = Sucrose Control; Vit = Vitamins C + E; Veh = Vehicle