Maternal ethanol (E) consumption often has a devastating and lasting effect on fetal growth and development, both in humans and in animal models. Yet, the mechanism(s) for this remaining uncertain and no treatment, either preventative or postinsult for the fetal damage has evolved. Previous studies in our laboratory have documented a variety of fetotoxic responses to E using rat models for in vivo exposure and primary cultures of fetal cells in vitro. Recently we reported that in cultured fetal rat hepatocytes (FRH), E blocked replication, induced total and mitochondrial membrane damage, increased production of reactive oxygen species [O2 and H2O2 (ROS)] and membrane lipid peroxidation products in whole cell and mitochondria (M), and reduced GSH pools and ATP synthesis. Importantly, normalizing cell antioxidant status could reverse these measures and restore cell replicative capacity. Preliminary studies included here illustrate similar responses in vivo after a maternal binge exposure as well as in cultured neonatal astrocytes (A) and cardiac myocytes (CM), and documented an E-related inhibition of M respiratory complexes I and IV. Also, administration of allopurinol (AL) to dams concomitant with E, normalized H2O2 production and GSH in fetal brain (but not liver), indicating a xanthine oxidase interaction. Thus, we hypothesize, first, that the underlying mechanism for the toxic effects of E on fetal cells is E-induced generation of ROS, second, that many of these fetotoxic effects can be prevented or mitigated by treatments that optimize oxidant defense systems in fetal tissues, and third, that E damage of M plays an important role in these processes. The models will be Sprague Dawley rats exposed to E from gestation days (GD) 3-20 (chronic) or on GD days 11-13, 14-17, 18-20 (binge). We will focus on fetal brain, heart and liver as known targets fore, and on key cells of these tissues, cultured RFH, A, and CM.
Specific Aim 1 is to define the oxidative stress with respect to ROS production, oxidative products in membrane lipids (MDA, dienes, 4-hydroxynonenal), proteins (thiol oxidation, aldehyde adducts), DNA (8-hydroxydeoxyguanosine), antioxidant status (GSH, vitamin E, ascorbate), and activities of SOD, catalase, and glutathione peroxidase. We will determine the rate of onset; duration, degree, and gestational timing of E exposure needed to elicit effects; and the reversibility of these parameters.
Specific Aim 2 will develop treatment regimens to prevent or reduce this oxidative stress and its consequences. To this end, we will optimize oxidative defenses in E-exposed fetal tissues by maintaining GSH stores (with N- acetylcysteine, S-adenosylmethionine) and other antioxidants (vitamin E, ascorbate), and by administration of a xanthine oxidase inhibitor (AL). These treatments will be tested individually and in combination to be tailored to specific E exposure patterns and tissue requirements.
Specific Aim 3 will test the hypothesis that there are close connections between E-related oxidative stress and M damage and that this is an important factor in the fetotoxicity of E. We will define effects of E on M integrity and function (especially respiratory chain) with respect to onset, exposure pattern, and reversal by antioxidant treatment.
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