Metabolism of the cytochrome P450 mechanism-based inhibitor N-benzyl-1-aminobenzotriazole to products that covalently bind with protein in guinea pig liver and lung microsomes: comparative study with 1-aminobenzotriazole.
The metabolism and covalent binding of radioactivity to microsomal protein of the cytochrome P450 (P450) mechanism-based inhibitors 1-amino-[14C]-2,3-benzotriazole ([14C]ABT) and two radiolabeled forms of N-benzyl-1-aminobenzotriazole (BBT), N-benzyl-1-amino-[14C]-2,3-benzotriazole ([14C]-2,3-BBT) and [14C]-N-7-benzyl-1-aminobenzotriazole ([14C]-7-BBT), were examined in hepatic or pulmonary microsomes from untreated and phenobarbital (PB)- or beta-naphthoflavone (betaNF)-induced guinea pigs. [14C]-2,3-BBT and [14C]-7-BBT were converted to multiple metabolites including ABT, benzotriazole, benzaldehyde, 2- or 3-hydroxy-BBT, and 4-hydroxy-BBT by hepatic microsomes, while [14C]ABT, whose primary metabolite was benzotriazole, underwent little biotransformation. Neither ABT nor BBT was extensively metabolized by pulmonary microsomes. Hepatic microsomes from betaNF (vs PB)-treated guinea pigs metabolized [14C]ABT, [14C]-2,3-BBT, and [14C]-7-BBT more extensively. The degree of NADPH-dependent covalent binding of [14C]-2,3-BBT- or [14C]-7-BBT-derived radioactivity (1.0 nmol/mg of protein) was higher than that of [14C]ABT (0.3-0.8 nmol/mg of protein) in hepatic microsomes, especially those from PB-induced animals. Covalent binding per nmol of P450 in pulmonary microsomes was 3-4-fold higher with [14C]-2,3-BBT (2.9 nmol/nmol of P450) than with [14C]-7-BBT (1.0 nmol/nmol of P450), whereas in hepatic microsomes from PB- or betaNF-treated animals the ratio of binding with the two forms of BBT was approximately 1:1. [14C]-2,3-BBT- and [14C]-7-BBT-derived radioactivity was covalently bound to proteins that migrated in the molecular weight region corresponding to P450 on SDS-PAGE following incubation with NADPH. These data indicate that BBT is metabolized to at least two reactive compounds capable of covalent modification of protein and/or a single reactive product is formed which contains both the benzo ring (of benzotriazole) and the benzyl carbon atom (of the N-benzyl group); that P450 apoprotein modification may be an important mechanism of inactivation of pulmonary and hepatic P450 by BBT; and that hepatic microsomes from betaNF-induced guinea pigs generate more metabolites that do not act as mechanism-based P450 inhibitors from BBT than do those from PB-induced animals.
Woodcroft
K J
KJ
Department of Pharmacology and Toxicology, University of Western Ontario, London, Canada.
Webb
C D
CD
Yao
M
M
Weedon
A C
AC
Bend
J R
JR
D000818:Animals; D001711:Biotransformation; D002250:Carbon Radioisotopes; D065607:Cytochrome P-450 Enzyme Inhibitors; D004789:Enzyme Activation / Q000187:drug effects; D008401:Gas Chromatography-Mass Spectrometry; D006168:Guinea Pigs; D008168:Lung / Q000201:enzymology*; D008297:Male; D008861:Microsomes / Q000201:enzymology / Q000378:metabolism*; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism*; D011485:Protein Binding; D014230:Triazoles / Q000493:pharmacokinetics*
D002250:Carbon Radioisotopes; D065607:Cytochrome P-450 Enzyme Inhibitors; D014230:Triazoles; C067013:N-benzyl-1-aminobenzotriazole; C033020:1-aminobenzotriazole