Cytochrome P450 catalyzed metabolism of 1,2-dibromoethane in liver microsomes of differentially induced rats

Chem Biol Interact. 1996 Jan 5;99(1-3):41-53. doi: 10.1016/0009-2797(95)03659-8.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetaldehyde / analogs & derivatives*
  • Acetaldehyde / metabolism
  • Adenosine / analogs & derivatives
  • Adenosine / metabolism
  • Animals
  • Benzoflavones / pharmacology
  • Chromatography, High Pressure Liquid
  • Cytochrome P-450 Enzyme System / metabolism*
  • Enzyme Induction / drug effects
  • Enzyme Induction / genetics
  • Enzyme Inhibitors / pharmacology
  • Ethylene Dibromide / metabolism*
  • Ethylene Dibromide / toxicity
  • Hydrogen-Ion Concentration
  • Hypnotics and Sedatives / pharmacology
  • Isoenzymes / drug effects
  • Isoenzymes / metabolism
  • Kinetics
  • Male
  • Microsomes, Liver / metabolism*
  • Molecular Structure
  • NADP / metabolism
  • NADP / pharmacology
  • Phenobarbital / pharmacology
  • Pyrazoles / pharmacology
  • Rats
  • Rats, Wistar
  • Temperature
  • beta-Naphthoflavone

Substances

  • Benzoflavones
  • Enzyme Inhibitors
  • Hypnotics and Sedatives
  • Isoenzymes
  • Pyrazoles
  • bromoacetaldehyde
  • Ethylene Dibromide
  • 1,N(6)-ethenoadenosine
  • pyrazole
  • NADP
  • beta-Naphthoflavone
  • Cytochrome P-450 Enzyme System
  • Acetaldehyde
  • Adenosine
  • Phenobarbital