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

article





Wormhoudt, L.W.

Ploemen, J.H.T.M.

Commandeur, J.N.M.

Ommen, B. van

Bladeren, P.J. van

Vermeulen, N.P.E.
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 (ε-ADO), which was measured by HPLC, After microsomal incubation with I,2-DBE in the presence of ADO and removal of proteins by denaturation and centrifugation, derivatization by heating 4 h at 65°C appeared necessary to ensure efficient formation of ε-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), β-naphtoflavone (βNF) and pyrazole (PYR), P450 isoenzymes in PYR- and βNF-induced microsomes showed linear enzyme kinetics while P450 isoenzymes in control and PB-induced microsomes showed non-linear enzyme kinetics. The apparent V(max)- and K(m)-values for the metabolism of 1,2-DBE to 2-BA were 2.5 nmol/min/mg protein and 144 μM for P450 isoenzymes in PYR-induced microsomes and 773 pmol/min/mg protein and 3.3 mM for P450 isoenzymes in βNF-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 V(max)- and K(m)-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 βNF-induced microsomes and thus much lower than the PYR-induced microsomes.
Chemicals/CAS: 1,N(6)-ethenoadenosine, 39007-51-7; Acetaldehyde, 75-07-0; Adenosine, 58-61-7; Benzoflavones; beta-Naphthoflavone, 6051-87-2; bromoacetaldehyde, 17157-48-1; Cytochrome P-450 Enzyme System, 9035-51-2; Enzyme Inhibitors; Ethylene Dibromide, 106-93-4; Hypnotics and Sedatives; Isoenzymes; NADP, 53-59-8; Phenobarbital, 50-06-6; pyrazole, 288-13-1; Pyrazoles
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1,2-Dibromoethane (ethylene dibromide)1,N6-ethanoadenosine2-BromoacetaldehydeCytochrome P450InductionRat liver microsomesAcetaldehydeAdenosineAnimalsBenzoflavonesbeta-NaphthoflavoneChromatography, High Pressure LiquidCytochrome P-450 Enzyme SystemEnzyme InductionEnzyme InhibitorsEthylene DibromideHydrogen-Ion ConcentrationHypnotics and SedativesIsoenzymesKineticsMaleMicrosomes, LiverMolecular StructureNADPPhenobarbitalPyrazolesRatsRats, WistarTemperature
TNO Identifier
233191
Repository link
https://resolver.tno.nl/uuid:3018b84b-1378-410c-b27d-261a977b20c7
ISSN
00092797
Source
Chemico-Biological Interactions, 99(1-3), pp. 41-53.
Pages
41-53
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