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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