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AMELIORATION OF ETHANOL-INDUCED DYSMORPHOGENESIS IN RODENT EMBRYOS IN VITRO USING ADENOVIRAL-MEDIATED EXPRESSION OF CU,ZN-SUPEROXIDE DISMUTASE (SOD1) OR MN-SUPEROXIDE DISMUTASE (SOD2)
Citation:
Hunter III, E S., J. B. Smith, M R. Blanton, P C. Hartig, AND K. K. Sulik. AMELIORATION OF ETHANOL-INDUCED DYSMORPHOGENESIS IN RODENT EMBRYOS IN VITRO USING ADENOVIRAL-MEDIATED EXPRESSION OF CU,ZN-SUPEROXIDE DISMUTASE (SOD1) OR MN-SUPEROXIDE DISMUTASE (SOD2). Presented at International Conference on Genes and Gene Therapy for Treatment of Alcohol Related Disease, Chapel Hill, NC, May 7-11, 2001.
Description:
Ethanol produces dysmorphogenesis in offspring when pregnant animals are exposed in vivo (Chaudhuri 2000; Hannigan and Armant, 2000) and by direct exposure of neurulation staged embryos to ethanol in vitro (Kotch et al., 1995; Hunter et al., 1994 ; Thompson and Folb, 1982). The abnormalities induced by ethanol are well characterized and most of the craniofacial defects are the result of effects on the neural crest cell population (Webster and Ritchie, 1991; Cartwright et al., 1998; Chen et al., 2000). Previous studies showed that explanted embryos exposed to ethanol in culture medium supplemented with bovine erythrocyte superoxide dismutase (SOD) had decreased malformations, lipid peroxides (i.e. thiobarbituric acid reactive moieties) and cell death (Kotch et al., 1995). Exogenous SOD also ameliorates malformations produced by other xenobiotics (Eriksson and Borg, 1991; Karabulut et al., 2000; Wells et al., 1997) and this suggests that reactive oxygen species are important mediators of dysmorphogenesis. Despite these studies that expose conceptuses to exogenous SOD, it remains to be determined whether the morphological protection results from an increased embryonic antioxidative capability. Therefore, we tested the hypothesis that expression of SOD in the embryo would ameliorate ethanol-induced defects. Additionally, we compared the protective effects of cytoplasmic Cu,Zn-superoxide dismutase (SOD1) and mitochondrial Mn-superoxide dismutase (SOD2) in our model (Lam et al., 1997). CD-1 strain neurulation-staged mouse conceptuses were prepared for whole embryo culture using standard techniques (Sadler, 1979). The UNC-CH vector core provided SOD1 and SOD2 expressing adenoviruses. Since the neural crest cells are a critical target cell population, we used an intramnionic route of adenoviral administration in embryo culture. Using this technique, expression of marker genes (lacZ and green fluorescence protein) in embryos showed high levels of expression in the neuroepithelium and its neural crest cell derivatives within 2-4 hours of administration (Hartig and Hunter, 1998). Non-toxic concentrations of adenoviruses were identified and utilized in subsequent studies. Direct exposure of mouse conceptuses to ethanol (108 mM) induced neural tube closure defects, prosencephalic and pharyngeal arch hypoplasia and heart outflow tract defects in embryo culture. Ethanol exposure also increased reactive oxygen intermediates (ROI) as monitored by formazan production from MTT. In embryos transduced with SOD2 and exposed to ethanol, there was a decrease in ethanol-induced prosencephalic and pharyngeal arch hypoplasia, heart outflow tract defects, MTT reactive ROI and prosencephalic TUNEL-positive cell death. In contrast, SOD1 expression reduced pharyngeal arch abnormalities but none of the other effects that were examined. No changes in ethanol-induced defects were observed in embryos transduced with lacZ. The differential protective effect of SOD1 and SOD2 indicates that mitochondria are critical target organelles in the embryo to ethanol-induced damage. Despite the amelioration of morphological effects, we were unable to demonstrate a corresponding increase in SOD1 or SOD2 protein by Western analysis in whole embryos. We know from studies of marker gene that adenoviral-mediated transduction is most robust in neural crest cells. Since SOD ameliorated craniofacial defects that resulted from damage to the neural crest cells and the neural crest cells constitute a relatively small percentage of the whole embryo, our ability to see morphological protection without increased SOD protein assessed by Westerns are consistent observations. Additionally, chick neural crest cells have no detectable SOD activity (Davis et al., 1990). Therefore, even limited expression of SOD in crest cells in our studies may impart protective effects without a detectable change in SOD in the whole embryo. We are continuing our studies to evaluate the direct effects of ethanol on the neural crest cells and the ability of adenoviral-mediated transduction of SOD to ameliorate the adverse effects of ethanol on those cells.