New Emerging Team on FASD: Oxidative Stress, Biomarkers & Antioxidant Therapy Leader: James F. Brien, Queen’s University Canadian Institutes of Health Research

Biomarkers B Antioxidant Therapy A Oxidative Stress C CIHR NEW EMERGING TEAM ON FASD

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 production of O 2 radicals Cell degradation of O 2 radicals Overabundance of O 2 radicals/Oxidative Stress

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 H 2 O 2 O 2 –  OH  Brain Injury of FASD

Measures of Oxidative Stress 1. Glutathione (GSH): Intracellular GSH localized primarily in mitochondria and cytoplasm. 2. F 2 -Isoprostanes: Prostaglandin F 2 -like compounds. Formed in vivo by nonenzymatic free radical- induced peroxidation of arachidonic acid. Specific and stable products of lipid peroxidation. 8-iso-Prostaglandin F 2 

HIPPOCAMPUS Ethanol Offspring Control Offspring

Experimental Animal Study Design Timed Pregnant Guinea Pigs EthanolIsocaloric-Sucrose/Water (4g/kg MBW/day) Pair-Feeding Term Fetus (GD 65) [GSH] in mitochondria and cytoplasm [8-iso-PGF 2  ] in homogenate Hippocampus

Fetal 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

CYTOCHROME C (FETAL HIPPOCAMPUS) EthanolSucrose

ETHANOL SUCROSE WATER ACTIVATED CASPASE-3 (FETAL HIPPOCAMPUS)

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-PGF 2  ].

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

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

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

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 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 Vitamins C + EVehicleVitamins C + EVehicle Nutritional Control Chronic Treatment Regimen (term, about GD 68; range, GD 66-70): Daily oral administration of vitamins C (250mg) + E (100mg) OR vehicle (milk/cream, 0/1, v/v). 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 0: Litters (n=3-4 littermates) born by spontaneous vaginal delivery. One littermate euthanized and brain weight determined. PD 45 (range, PD 43-47): Morris water-maze task for spatial learning and memory. Moving-platform paradigm with location of hidden platform changed to a new quadrant every second day; “new locations” versus “old locations” over eight consecutive days. Daily testing consisted of two blocks of two trials each, with 5-min period between each block. 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 + EVehicleVitamins C + EVehicle Nutritional Control Ethanol Vitamins C + EVehicleVitamins C + EVehicle Nutritional Control Ethanol Vitamins C + EVehicleVitamins C + EVehicle Nutritional Control Ethanol Vitamins C + E VehicleVitamins C + EVehicle Nutritional Control

MORRIS WATER MAZE

Figure 1: PD0 brain and hippocampal weights. Ethanol decreased brain weight compared with control; Vitamin C plus E treatment protected hippocampal weight in ethanol offspring. E = Ethanol; S = Sucrose Nutritional Control; Vit = Vitamins C + E; Veh = Vehicle Group means with different letters are statistically different (p < 0.05) Figure 1: PD0 brain and hippocampal weights. Ethanol decreased brain weight compared with control; Vitamin C plus E treatment protected hippocampal weight in ethanol offspring. E = Ethanol; S = Sucrose Nutritional Control; Vit = Vitamins C + E; Veh = Vehicle Group means with different letters are statistically different (p < 0.05) Figure 1: PD0 brain and hippocampal weights. Ethanol decreased brain weight compared with control; Vitamin C plus E treatment protected hippocampal weight in ethanol offspring. E = Ethanol; S = Sucrose Nutritional Control; Vit = Vitamins C + E; Veh = Vehicle Group means with different letters are statistically different (p < 0.05) Figure 1: PD0 brain and hippocampal weights. Ethanol decreased brain weight compared with control; Vitamin C plus E treatment protected hippocampal weight in ethanol offspring. E = Ethanol; S = Sucrose Nutritional Control; Vit = Vitamins C + E; Veh = Vehicle Group means with different letters are statistically different (p < 0.05) Figure 1: PD0 brain and hippocampal weights. Ethanol decreased brain weight compared with control; Vitamin C plus E treatment protected hippocampal weight in ethanol offspring. E = Ethanol; S = Sucrose Nutritional Control; Vit = Vitamins C + E; Veh = Vehicle Group means with different letters are statistically different (p < 0.05) Figure 1: PD0 brain and hippocampal weights. Ethanol decreased brain weight compared with control; Vitamin C plus E treatment protected hippocampal weight in ethanol offspring. E = Ethanol; S = Sucrose Nutritional Control; Vit = Vitamins C + E; Veh = Vehicle Group means with different letters are statistically different (p < 0.05) Figure 1: PD0 brain and hippocampal weights. Ethanol decreased brain weight compared with control; Vitamin C plus E treatment protected hippocampal weight in ethanol offspring. E = Ethanol; S = Sucrose Nutritional Control; Vit = Vitamins C + E; Veh = Vehicle Group means with different letters are statistically different (p < 0.05) Figure 1: PD0 brain and hippocampal weights. Ethanol decreased brain weight compared with control; Vitamin C plus E treatment protected hippocampal weight in ethanol offspring. E = Ethanol; S = Sucrose Nutritional Control; Vit = Vitamins C + E; Veh = Vehicle Group means with different letters are statistically different (p < 0.05) PD0: Brain and hippocampal weights Ethanol decreased brain weight compared with control; Vitamin C plus E treatment protected hippocampal weight in ethanol offspring. Figure 1: PD0 brain and hippocampal weights. Ethanol decreased brain weight compared with control; Vitamin C plus E treatment protected hippocampal weight in ethanol offspring. E = Ethanol; S = Sucrose Nutritional Control; Vit = Vitamins C + E; Veh = Vehicle Group means with different letters are statistically different (p < 0.05) 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. E = Ethanol; S = Sucrose Control; Vit = Vitamins C + E; Veh = Vehicle

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. SUMMARY

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

Biomarkers B Antioxidant Therapy Training D A Oxidative Stress C CIHR NEW EMERGING TEAM ON FASD PDF PhD MSc

R. Cohen-Kerem & G. Koren, Neurotoxicol. Teratol. (2003). Ethanol- induced Oxidative Stress Mechanism

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

ETHANOLSUCROSEWATER 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. E = Ethanol; S = Sucrose Control; Vit = Vitamins C + E; Veh = Vehicle