New Emerging Team on FASD: Oxidative Stress, Biomarkers & Antioxidant Therapy

New Emerging Team on FASD:Oxidative Stress, Biomarkers & Antioxidant Therapy

New Emerging Team on FASD:Oxidative Stress, Biomarkers & Antioxidant Therapy

Leader: James F. Brien, Queen’s UniversityLeader: James F. Brien, Queen’s University

Canadian Institutes of Health ResearchCanadian Institutes of Health Research

Biomarkers

B

Antioxidant Therapy

AOxidative Stress

C

CIHR NEW EMERGING TEAM ON FASDCIHR NEW EMERGING TEAM ON FASD

Members of NET on FASDMembers of NET on FASDAlan 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 OBJECTIVESRESEARCH 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 BrainCoronal Section of the Brain





OBJECTIVE AOBJECTIVE ATo determine whether oxidative stress is a

mechanism of the brain injury of FASD.

Definition of Oxidative StressDefinition of Oxidative StressOxygen radicals: highly reactive molecules

generated during cell metabolism.

Cell production

of O2 radicals

Cell degradation

of O2 radicals

Overabundance of O2 radicals/Oxidative Stress

Proposed Mechanism of Brain Injury of FASD

Proposed Mechanism of Brain Injury of FASD

Maternal Ingestion of Ethanol

Fetal Brain Exposure to Ethanol

Damage to Key Cell Molecules(DNA, Proteins, Membrane Phospholipids)

Neuronal Cell Death

Oxidative Stress / Increased Reactive Oxygen Species

H2O2 O2– OH

Brain Injury of FASD

Measures of Oxidative StressMeasures 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 radical-induced peroxidation of

arachidonic acid.• Specific and stable products of lipid peroxidation.

8-iso-Prostaglandin F2

HIPPOCAMPUS

Ethanol Offspring

Control Offspring

Experimental Animal Study DesignExperimental Animal Study DesignTimed Pregnant Guinea Pigs

Ethanol Isocaloric-Sucrose/ Water(4g/kg MBW/day) Pair-Feeding

Term Fetus (GD 65)

[GSH] in mitochondria and cytoplasm

[8-iso-PGF2] in homogenate

Hippocampus

Fetal HippocampusFetal Hippocampus

MITOCHONDRIA

Ethanol Sucrose Water0.0

2.5

5.0

a a

b

TREATMENT

[GS

H]

(mm

ol/

mg

pro

tein

)

CYTOSOL

Ethanol Sucrose Water0

10

20

30

40

50

60

TREATMENT

[GS

H]

(mm

ol/

mg

pro

tein

)

GLUTATHIONE


50

100

150

200

250GD 65PD 0

TREATMENT

[8-i

so-P

GF

2

](p

g/m

g p

rote

in)

Fetal HippocampusFetal Hippocampus

8-iso-PGF2

G. Weaver, University of Colorado at Denver

Modified from J.E. Dawson and L.M Winn

Apoptosis (Programmed Cell Death) and Caspase-3

Apoptosis (Programmed Cell Death) and Caspase-3

CYTOCHROME CYTOCHROME CC (FETAL HIPPOCAMPUS) (FETAL HIPPOCAMPUS)

Ethanol Sucrose


10

20

30 *

TREATMENT

AC

TIV

AT

ED

CA

SP

AS

E-3

IMM

UN

OR

EA

CT

IVIT

Y(%

of

all

Ce

lls

)

ETHANOL SUCROSE WATER

ACTIVATED CASPASE-3 (FETAL HIPPOCAMPUS)

SUMMARYSUMMARY

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].

Disruption of the mitochondria and consequent apoptosis play key roles in the mechanism of the brain injury of FASD involving the hippocampus.

CONCLUSIONCONCLUSION





EthanolSucrose

INDIVIDUAL FAEE

3.0

2.0

1.0

0ME

CO

NIU

M I

ND

IVID

UA

L [

FA

EE

]

(

nm

ol/

g)

Individual [FAEE] in meconium of term fetal offspring of the ethanol and isocaloric-sucrose/pair-fed groups

Ethanol Sucrose Water

*4.0

3.0

2.0

1.0

0ME

CO

NIU

M T

OT

AL

[F

AE

Es]

(n

mo

l/g

)

TREATMENT

Total [FAEEs] in meconium of term fetal offspring of the ethanol, isocaloric-sucrose/pair-fed and water groups





Rationale for Vitamin C + Vitamin E StudyRationale 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).

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.

Ethanol

Vitamins C + E Vehicle Vitamins C + E Vehicle

Nutritional ControlEthanol

Vitamins C + E Vehicle Vitamins C + E Vehicle

Nutritional Control

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.

E +Vit E + Veh S + Vit S + Veh0.0

0.5

1.0

1.5

2.0

2.5

a ab b

Treatment

Bra

in W

eig

ht

(g)

E + Vit E + Veh S + Vit S + Veh0.000

0.025

0.050

0.075

0.100

0.125

ab b b

Treatment

Hip

po

cam

pu

s W

eig

ht/

Bra

in W

eig

ht

Rat

io

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).

Vitamins C + E produced deficits in both acquisition (New Locations) and retention (Old Locations) of new memory in control offspring.

"New Locations" of Platform

E + Vit E + Veh S + Vit S + Veh0

10

20

30

ba

aa

TREATMENT

AV

ER

AG

E T

IME

RE

QU

IRE

D T

OL

OC

AT

E H

IDD

EN

PL

AT

FO

RM

(s) "Old Locations" of Platform


5

10

15

20

25

ad

b,ca,c

TREATMENT

AV

ER

AG

E T

IME

RE

QU

IRE

D T

OL

OC

AT

E H

IDD

EN

PL

AT

FO

RM

(S

)


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.

SUMMARYSUMMARY

Vitamins C + E dose studies are being conducted to determine optimal antioxidant vitamin therapy for the brain injury of FASD.

CONCLUSIONCONCLUSION

Biomarkers

B

Antioxidant Therapy

Training

D

AOxidative Stress

C

CIHR NEW EMERGING TEAM ON FASDCIHR NEW EMERGING TEAM ON FASD

PDF

PhD

MSc

R. Cohen-Kerem & G. Koren, Neurotoxicol. Teratol. (2003).

Ethanol-induced Oxidative Stress MechanismEthanol-induced Oxidative Stress Mechanism

HIPPOCAMPAL CA1 PYRAMIDAL CELL LOSSHIPPOCAMPAL 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


10

20

30 *

TREATMENT

CL

EA

VE

D P

AR

PIM

MU

NO

RE

AC

TIV

ITY

(% o

f a

ll C

ells

)

ETHANOL SUCROSE WATER

CLEAVED PARP (FETAL HIPPOCAMPUS)

PD 45: Morris water maze

Ethanol and/or vitamins C + E treatment did not affect swim speed in locating the hidden platform.

"New Locations" of Platform


50

100

150

200

250

TREATMENT

SW

IM S

PE

ED

IN

LO

CA

TIN

GH

IDD

EN

PL

AT

FO

RM

(cm

/s)

"Old Locations" of Platform


50

100

150

200

250

TREATMENTS

WIM

SP

EE

D I

N L

OC

AT

ING

HID

DE

N P

LA

TF

OR

M (

cm/s

)


Documents

New Emerging Team on FASD: Oxidative Stress, Biomarkers & Antioxidant Therapy