title: Cytochrome P450 catalyzed metabolism of 1,2-dibromoethane in liver microsomes of differentially induced rats.
abstract: The cytochrome P450 (P450) catalyzed oxidation of 1,2-dibromoethane (1,2-DBE) to 2-bromoacetaldehyde (2-BA) was measured in liver microsomes of both control and differentially induced rats. 2-BA formation was quantified by derivatization of 2-BA with adenosine (ADO), resulting in the formation of the highly fluorescent 1,N6-ethenoadenosine (epsilon-ADO), which was measured by HPLC. After microsomal incubation with 1,2DBE in the presence of ADO and removal of proteins by denaturation and centrifugation, derivatization by heating 4 h at 65 degrees C appeared necessary to ensure efficient formation of epsilon-ADO. Using this optimized derivatization method to quantitate 2-BA formation, the enzyme kinetics of the P450 catalyzed oxidation of 1,2-DBE to 2-BA were measured in liver microsomes prepared from untreated rats and rats pretreated with phenobarbital (PB), beta-naphtoflavone (beta NF) and pyrazole (PYR). P450 isoenzymes in PYR- and beta NF-induced microsomes showed linear enzyme kinetics while P450 isoenzymes in control and PB-induced microsomes showed non-linear enzyme kinetics. The apparent Vmax- and Km- values for the metabolism of 1,2-DBE to 2-BA were 2.5 nmol/min/mg protein and 144 microns for P450 isoenzymes in PYR-induced microsomes and 773 pmol/min/mg protein and 3.3 mM for P450 isoenzymes in beta NF-induced microsomes, respectively. Due to the non-linear enzyme kinetics of the P450 catalyzed oxidation of 1,2-DBE to 2-BA using control and PB-induced microsomes, no proper Vmax- and Km- values could be calculated. However, from Michaelis-Menten plots it was clear that the affinity of P450 isoenzymes for 1,2-DBE in control and PB-induced microsomes was in the same range when compared to beta NF-induced microsomes and thus much lower than the PYR-induced microsomes.
authors.affiliation: Leiden Amsterdam Center for Drug Research (LACDR), Vrije Universiteit, Department of Pharmacochemistry, The Netherlands.
authors.forename: L W
authors.initials: LW
authors.lastname: Wormhoudt
authors.forename: J H
authors.initials: JH
authors.lastname: Ploemen
authors.forename: J N
authors.initials: JN
authors.lastname: Commandeur
authors.forename: B
authors.initials: B
authors.lastname: van Ommen
authors.forename: P
authors.initials: P
authors.lastname: van Bladeren
authors.forename: N P
authors.initials: NP
authors.lastname: Vermeulen
mesh_terms: D000079:Acetaldehyde / Q000031:analogs & derivatives* / Q000378:metabolism; D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001571:Benzoflavones / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004790:Enzyme Induction / Q000187:drug effects / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015946:Ethylene Dibromide / Q000378:metabolism* / Q000633:toxicity; D006863:Hydrogen-Ion Concentration; D006993:Hypnotics and Sedatives / Q000494:pharmacology; D007527:Isoenzymes / Q000187:drug effects / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism*; D015394:Molecular Structure; D009249:NADP / Q000378:metabolism / Q000494:pharmacology; D010634:Phenobarbital / Q000494:pharmacology; D011720:Pyrazoles / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013696:Temperature; D019324:beta-Naphthoflavone
chemical_list: D001571:Benzoflavones; D004791:Enzyme Inhibitors; D006993:Hypnotics and Sedatives; D007527:Isoenzymes; D011720:Pyrazoles; C038235:bromoacetaldehyde; D015946:Ethylene Dibromide; C007640:1,N(6)-ethenoadenosine; C031280:pyrazole; D009249:NADP; D019324:beta-Naphthoflavone; D003577:Cytochrome P-450 Enzyme System; D000079:Acetaldehyde; D000241:Adenosine; D010634:Phenobarbital