Covalent binding of ethylene dibromide and its metabolites to albumin.
The present study was undertaken to determine covalent binding of [1,2-14C]ethylene dibromide (EDB) to albumin under in vivo and in vitro conditions. For the in vivo covalent binding, 25 mg/kg body weight of [1,2-14C]EDB was given daily to male rats for 12 consecutive days and the animals were sacrificed at 24 h following the last dose. Blood was withdrawn from inferior vena cava in heparinized tubes and plasma was separated, dialyzed against ice-cold 10 mM phosphate buffer (pH 7.4) and then subjected to size-exclusion high-performance liquid chromatography (SE-HPLC). A major radioactive peak eluted at an elution volume corresponding to 65,000 dalton molecular mass was found to be associated to albumin at a level of 0.14 nmol equivalent EDB/mg protein. For the in vitro covalent binding, human plasma or purified albumin was incubated with [1,2-14C]EDB in the presence of phenobarbital-treated rat liver microsomes and NADPH-generating system for 2 h at 37 degrees C. The 100,000 x g supernatant of the incubation mixture was dialyzed extensively and analyzed as described for the in vivo studies. Approximately 0.28 nmol equivalent EDB/mg protein was found to be associated to albumin (about 2-fold higher than the in vivo binding). Binding of 14C-label to albumin under in vivo and in vitro conditions was further supported by the affinity chromatography of albumin fraction isolated by SE-HPLC. Reversed-phase HPLC analysis of pronase digest of the albumin obtained from in vitro studies indicated formation of several amino acid adducts of EDB and/or its metabolites. Structure elucidation of such amino acid adducts will be helpful in developing a relatively non-invasive method of measuring the EDB exposure.
Kaphalia
B S
BS
Department of Pathology, University of Texas Medical Branch, Galveston 77555-0605.
Ansari
G A
GA
D000284:Administration, Oral; D000818:Animals; D001798:Blood Proteins / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D015946:Ethylene Dibromide / Q000378:metabolism*; D006801:Humans; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012709:Serum Albumin / Q000378:metabolism*
D001798:Blood Proteins; D012709:Serum Albumin; D015946:Ethylene Dibromide