ABSTRACT: Specific gene changes after exposure to the heart toxin, bis(2-chloroethoxy)methane, were analyzed to elucidate critical pathways associated with recovery from heart toxicity. Bis(2-chloroethoxy)methane was administered to male F344/N rats at 0, 200, 400, or 600 mg/kg by dermal exposure for up to 16 days. Heart toxicity occurred at day 2 of exposure in all three regions of the heart (atrium, ventricle, interventricular septum) and was characterized by myofiber vacuolation, necrosis, mononuclear-cell infiltration, and atrial thrombosis. Ultrastructural analysis revealed that the primary site of damage occured in the mitochondrion. Damaged mitochondria had disrupted cristae and loss of membrane structure, and distention of the myocardial sarcoplasmic reticulum. By day 5, even though daily dosing was continued, the heart toxic lesions begin to resolved. By day 16, even though BCEM treatment continued, the heart appeared normal. RNA was extracted from control and dosed rats at day 2 and day 5, and analyzed for gene change by PCR. Gene changes associated with promoting cell growth including up regulation of growth factors and cell adhesion molecules were up regulated. Temperature regulation was promoted by up regulation of uncoupling proteins. Genes controlling the action potential of the heart, including ion flow and G protein signals, were altered. Because mitochondria are damaged by BCEM exposure, preserving and restoring energy function to the heart is considered to be essential, and up regulation of components of the ATP synthase unit occurred by day 5. Significant gene changes included up regulation of transcription factors, growth factors, membrane bound G protein signals, and ion transport controls. Multiple gene changes in the heart may contribute to restoration of heart function after exposure to environmental cardiotoxins. Experiment Overall Design: Chemical and Animal exposures: Experiment Overall Design: Bis(2-chloroethoxy)methane (CAS No. 11-91-1; lot B004160277) (Karl Industries, Aurora, OH) (Fig, 1) was found to be 98.5% pure (Dunnick et al. 2004a; Dunnick et al. 2004b) . Solutions of BCEM were prepared in 95% ethanol for dosing by dermal administration daily to male F344 rats, excluding weekends, for two weeks plus two consecutive dosages before the last sacrifice on study-day 16, at which animals had received a total of 12 doses. Fur from the site of application was clipped weekly. Stock solutions prepared at concentrations of 0, 800, and 1200 mg/ml were stored in amber glass bottles at room temperature. The administrations were applied to the skin of the male rats at 0.5 ml/kg body to deliver doses of 0, 400, or 600 mg/kg body weight. All dose formulations were determined to be within ï±10% of target concentrations. Approximately 45% of a dermal dose of CEM is adsorbed (NIEHS Contract NO1-ES-75407 2002). Male F344/N rats (Taconic Laboratories, Germantown, NY) were placed on study at 5 weeks of age and housed one per cage in polycarbonate cages in rooms maintained at temperatures between 69 and 75ï°F with 35 â 65% relative humidity and a 12-hr light/dark cycle. Control and treated groups received irradiated NTP-2000 diet (Zeigler Brothers, Gardners, PA) ad libitum. Experiment Overall Design: Histopathology: Experiment Overall Design: Animals were anesthetized with CO 2 and heart collected and preserved in 10% formalin. Slides were made from paraffin sections from rats treated with BCEM as previously described (Dunnick et al. 2004a). Pathologic analysis included comparing heart lesions in male rats after 2, 3, 5, and 16 days of treatment. Experiment Overall Design: RNA Extraction Methods Experiment Overall Design: RNA was extracted from hearts of three control animal at day 2, three control rats at day 5, three rats exposed to 200, 400, or 600 mg/kg BCEM at day 2 and three at day 5. Animals designated for heart RNA extraction were anesthetized with CO2/O2 on study days 2 and 5, exanguinated, and their hearts infused with RNAlater. Total cardiac RNA was isolated from hearts using the QIAGEN Rneasy kit (QIAGEN, Valencia, CA). The RNA was quantified through optical density measurements and agarose gel electrophoresis, and kept frozen at â 70 C. RNA was extracted from control, 200, 400, and 600 mg/kg rats.
