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

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

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

3. 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.

5. RESEARCH OBJECTIVES • 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.

6. Coronal Section of the Brain

7. RESEARCH OBJECTIVES • 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.

8. 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 of O2 radicals Cell production of O2 radicals Overabundance of O2 radicals/Oxidative Stress

9. 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

10. 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 radical-induced peroxidation of arachidonic acid. • Specific and stable products of lipid peroxidation. 8-iso-Prostaglandin F2

12. HIPPOCAMPUS Control Offspring Ethanol Offspring

13. Experimental Animal Study Design Timed Pregnant Guinea Pigs Ethanol Isocaloric-Sucrose/ Water (4g/kg MBW/day) Pair-Feeding Term Fetus (GD 65) Hippocampus [GSH] in mitochondria and cytoplasm [8-iso-PGF2] in homogenate

14. Fetal Hippocampus

15. Fetal Hippocampus 8-iso-PGF2

16. G. Weaver, University of Colorado at Denver

17. Apoptosis (Programmed Cell Death) and Caspase-3 Modified from J.E. Dawson and L.M Winn

18. CYTOCHROME C (FETAL HIPPOCAMPUS) Ethanol Sucrose

19. ACTIVATED CASPASE-3 (FETAL HIPPOCAMPUS) ETHANOL SUCROSE WATER

20. 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].

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

22. RESEARCH OBJECTIVES • To test the hypothesis that oxidative stress is an important mechanism of the brain injury of FASD; B. To identify and validate reliable biomarkersfor 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.

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

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

25. RESEARCH OBJECTIVES • 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.

26. 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).

27. Ethanol Ethanol Ethanol Nutritional Control Nutritional Control Nutritional Control Vitamins C + E Vitamins C + E Vitamins C + E Vehicle Vehicle Vehicle Vitamins C + E Vitamins C + E Vitamins C + E Vehicle Vehicle Vehicle Ethanol NutritionalControl Ethanol Nutritional Control Vehicle Vitamins C + E Vehicle Vitamins C + E Vitamins C + E Vehicle Vitamins C + E 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. • 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.

28. MORRIS WATER MAZE

29. 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. 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. 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. 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. 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. 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. 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. 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. 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 Nutritional Control; Vit = Vitamins C + E; Veh = Vehicle Group means with different letters are statistically different (p < 0.05) 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 Nutritional Control; Vit = Vitamins C + E; Veh = Vehicle Group means with different letters are statistically different (p < 0.05) 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 Nutritional Control; Vit = Vitamins C + E; Veh = Vehicle Group means with different letters are statistically different (p < 0.05) 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 Nutritional Control; Vit = Vitamins C + E; Veh = Vehicle Group means with different letters are statistically different (p < 0.05) 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. E = Ethanol; S = Sucrose Control; Vit = Vitamins C + E; Veh = Vehicle

30. 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

31. 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.

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

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

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

35. 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

36. CLEAVED PARP (FETAL HIPPOCAMPUS) ETHANOL SUCROSE WATER

37. 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