Covalent binding of ethylene dibromide and its metabolites to albumin

Abstract

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 Blood was withdrawn from inferior vena cava in heparinized tubes and plasma was separated, dialyzed against ice-cold 10 mM phosphate buffer (pH 7.4) and then subjected to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°C. The 100 000 × 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.