1. FACE, Sep 20021 Antioxidants and Fetal Protection Against Ethanol Teratogenicity. Review of Experimental Data Raanan Cohen-Kerem Gideon Koren

2. FACE, Sep 20022 Outline Introduction Alcohol as an oxidant Studies review ­ Cell culture ­ Animal models Conclusions

3. FACE, Sep 20023 Introduction Heavy drinking during pregnancy can result in serious adverse outcomes for the fetus Fetal Alcohol Syndrome (FAS) ­ pre and/or postnatal growth retardation ­ central nervous system damage ­ typical facial dysmorphology Incidence of FAS - 0.97/1000 a year 50% of women in childbearing age consume alcohol

4. FACE, Sep 20024 Introduction The fetus is more susceptible to ethanol than the mother Ethanol affects biochemical processes and cellular structures through various mechanisms and most of them are still far from being completely understood

5. FACE, Sep 20025 Mechanisms of ethanol induced damage Amino acid activated ion channels GABANMDA Hypoxia FAEE Retinoic acid L1 neural cell adhesion molecule Endocrine Oxidative stress

6. FACE, Sep 20026 Objective To review the data of antioxidant effects in experimental models of fetal alcohol syndrome Treatment with antioxidants in pregnant alcoholic women may potentially prevent ethanol teratogenicity

7. FACE, Sep 20027 Indirect Direct C 2 H 5.OH (Ethanol) Glutathione pool Glutathione peroxidase activity Mitochondrial Damage Peroxidation of lipids, nucleic acids, and proteins Reactive oxygen species (ROS) Mitochondria CH3-CH-OH (Hydroxyethyl) (Hydroxyl) + + + - - + How does oxidative stress occur? OH + CYP2E1

8. FACE, Sep 20028 Antioxidants Endogenous ־ superoxide dismutase ־ glutathione peroxidase ־ catalase Exogenous ־ Vitamins Vitamin C Tocopherols  -Carotene Folic acid

9. FACE, Sep 20029 Literature Search Databases: ־MEDLINE ־EMBASE Keywords: pregnancy, ethanol, fetus, antioxidants, animal Included were reports on pregnancy outcome of cell culture or animal FAS model supplemented with antioxidants

10. FACE, Sep 200210 In the lab

11. FACE, Sep 200211 Vitamin E and beta-carotene protect against ethanol combined with ischemia in an embryonic rat hippocampal culture model of FAS Mitchell, Neurosci Lett 263:1999 ; Alcohol 17:1999. Reduced levels of cytochrome c oxidase mRNA Kim et al, 2001 Dose-dependent decreased viability of hippocampal cell cultures

12. FACE, Sep 200212 Nutritional supplementation ־ Vitamin E ־  -Carotene Limitations ־ Oxidative stress measures ־ Cells were exposed to ethanol in culture but not in vivo Vitamin E and beta-carotene protect against ethanol combined with ischemia in an embryonic rat hippocampal culture model of FAS Mitchell, Neurosci Lett 263:1999 ; Alcohol 17:1999.

13. FACE, Sep 200213 Effect of ethanol on rat fetal hepatocytes: studies on cell replication, lipid peroxidation and glutathione Devi, Hepatology18:1993 2.0mg/ml ethanol (~1/10 of Mitchell’s study) Outcome measure: cell replication Glutathione Antioxidants: ־ N-acetylcysteine ־ S-adenosylmethionine ־ Vitamin E

14. FACE, Sep 200214 In the lab

15. FACE, Sep 200215 FAS in rats: conditions for improvement of ethanol effects on fetal cerebral development with supplementary agents Tanaka, Biol Neonate 54:1988. Pregnant rats were fed 20% ethanol +0.03% vit. E, or 10% ethanol + 0.02% vit. E Outcome measures: ־ body ־ cerebral weights The investigation failed to show protective effect of vit. E

16. FACE, Sep 200216 Protective effects of the flavonoid mixture, silymarin, on fetal rat brain and liver La Grange, J Ethnopharmacol 65:1999. Antioxidant: Flavonoids (sylibum marianum) ­ Silymarin-Phytosome  Outcome measure ­ Tissue GGT activity in embryonic liver and brain

17. FACE, Sep 200217 Limitation ­ No oxidative stress measures to correlate Protective effects of the flavonoid mixture, silymarin, on fetal rat brain and liver La Grange, J Ethnopharmacol 65:1999.

18. FACE, Sep 200218 Fetoprotectivity of the flavanolignan compound siliphos against ethanol-induced toxicity Edwards, Phytother Res 14 :2000. Antioxidant: Sylibin Outcome measures ­ Fetal survival ­ GGT activity in the embryonic brain and liver ­ Glutathione (GSH) activity in the embryonic brain and liver

19. FACE, Sep 200219 Prevention by a silymarin/phospholipid compound of ethanol-induced social learning deficits in rats Reid, Planta Med 65:1999. Antioxidant: Silymarin Outcome measure ­ social recognition score of the offspring was obtained in two periods of 5 minutes separated by 60 minutes interval Limitations ­ No oxidative stress measures to correlate

20. FACE, Sep 200220 Protective effect of folic acid against oxidative stress produced in 21- day postpartum rats by maternal-ethanol chronic consumption during pregnancy and lactation period Cano, Free Radic Res 34:2001. Antioxidant: Folic acid 152  g/day Outcome measures ­ Body weight ­ Glutathione reductase in the fetal liver & pancreas ­ Carbonyl group proteins Fetal Body Weight

21. FACE, Sep 200221 Protective effect of folic acid against oxidative stress produced in 21- day postpartum rats by maternal-ethanol chronic consumption during pregnancy and lactation period Cano, Free Radic Res 34:2001. Fetal glutathione reductase Fetal carbonyl groups *-ethanol vs. control and vs.ethanol+FA

22. FACE, Sep 200222

23. FACE, Sep 200223 Alpha-tocopherol and gamma-tocopherol attenuate ethanol- induced changes in membrane fatty acid composition in embryonic chick brains Miller, Teratology 62 :2000. Injection of ethanol with or without vitamin E Outcome measure: ­ long chain membrane fatty acids in brain tissue ­ Brain mass

24. FACE, Sep 200224 Alpha-tocopherol and gamma-tocopherol attenuate ethanol- induced changes in membrane fatty acid composition in embryonic chick brains Miller, Teratology 62 :2000. Reduction in stearic acid, oleic acid, linoleic acid, linolenic acid and arachidonic acid Reduction in brain mass Supplementation of vitamin E prevented these effects and maintained brain mass

25. FACE, Sep 200225 Discussion A protective effect of antioxidants against ethanol-induced damage is observed in the available animal studies Can we apply this prophylactic treatment to pregnant women?

26. FACE, Sep 200226 Discussion Applying antioxidant treatment in pregnancy is not a new concept ­ Antioxidants (Vitamins E & C) for pre-eclampsia ­ These antioxidants were not found to be teratogenic Lancet 1999; 354: 810-16

27. FACE, Sep 200227 Conclusions Oxidative stress is a principal mechanism of ethanol induced damage Ethanol induced fetotoxicity was shown to be reduced by antioxidants in cell cultures and animal models Data regarding these effects in human are not available yet

28. FACE, Sep 200228 Take home message Treating women who abuse alcohol during pregnancy with antioxidants by food supplements as vitamins is appealing: ­ It may hold a protective effect against FAS as shown in the reviewed studies ­ it may reverse other nutritional deficits common in this population