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HALOACETIC ACIDS PERTURB PROTEIN PHOSPHORYLATION IN MOUSE EMBRYOS IN VITRO
Citation:
Blanton, M R. AND E S. Hunter III. HALOACETIC ACIDS PERTURB PROTEIN PHOSPHORYLATION IN MOUSE EMBRYOS IN VITRO. Presented at Society of Toxicology, Nashville, TN, March 17-21, 2002.
Description:
HALOACETIC ACIDS PERTURB PROTEIN PHOSPHORYLATION IN MOUSE EMBRYOS IN VITRO. MR Blanton and ES Hunter. Reproductive Toxicology Division, NHEERL, ORD, US EPA, RTP, NC, USA.
Sponsor: JM Rogers.
Haloacetic Acids (HAAs) formed during the disinfection process are present in drinking water. In vitro exposure of rodent conceptuses to these agents produces dysmorphology including craniofacial defects. Based on the effects of HAAs in adult tissues, we tested the hypothesis that exposure to HAAs perturbs signal transduction pathways. CD-1 mouse conceptuses (3-6 somites) were exposed to HAAs at concentrations that produce 100% dysmorphology [11mM dichloroacetate (DCA), 350?M dibromoacetate (DBA) or 300 ?M bromochloroacetate (BCA)] in whole embryo culture. To determine if exposure to the HAAs perturbs protein kinase activity, embryos were radiolabelled with 33-P-phosphate in HAA-containing or control medium during 0-2, 4-6 or 22-24 hours of exposure. 2D gel electrophoresis was then performed and autoradiographs compared using Phoretix 2D Software (Nonlinear Dynamics). Both time and chemical changes in protein phosphorylation were observed. To further evaluate protein phosphorylation, Western analysis on 1D gels was conducted using a phospho-tyrosine antibody (PY-99: Santa Cruz Biotechnology). Two major phosphotyrosine containing proteins (122 and 183kDa) were found to be affected by HAA exposure. No changes were observed as early as 2 hours after treatment but an increase in the 122kDa protein was found 6 hours following DCA exposure. By 24 hour after culture both phosphoproteins were increased in all three HAA treated groups with DCA producing the greatest effect. These changes indicate that the HAAs perturb signal transduction by altering the phosphorylation state of phospho-tyrosine proteins. Since pharmacological protein kinase inhibitors also produce dysmorphology, HAA-induced alteration of signal transduction may be responsible for altered differentiation and development. This abstract does not present EPA policy.