Cultured cells were treated with different concentrations of EtOH (20 or 50 mM) for 24 or 48 hrs. These EtOH concentrations were chosen for their physiological relevance, 20 mM is equivalent to DUI level and 50 mM falls within levels measured in alcoholics (Jones and Sternebring, 1992). The 24 or 48 hrs exposure times were chosen to mimic the likely prolonged exposure to maternal EtOH, based on the poor EtOH elimination capacity of the fetus during early development (Pikkarainen, 1971). Calculated volume of absolute EtOH (Fisher Scientific, Pittsburg, PA) was diluted in culture media and cells were fed daily with fresh media. Analysis was done in biological duplicates. In some experiments, cells were treated with 1 μM of 5-azacytidine (Sigma, St. Louis, MO) simultaneously with 20 mM EtOH for 24 hrs.
