Bioactivation of 8-methoxypsoralen and irreversible inactivation of cytochrome P-450 in mouse liver microsomes: modification by monoclonal antibodies, inhibition of drug metabolism and distribution of covalent adducts.
The in vitro bioactivation of 8-MOP was studied in liver microsomes of male CD-1 mice. In 10-min incubations with 40 microM [14C]8-MOP, covalent binding (mean +/- S.D.) was 1.8 +/- 0.4, 3.1 +/- 0.6 and 5.4 +/- 0.4 nmol/mg protein, respectively, in microsomes from mice pretreated for 3 days with vehicle, phenobarbital or beta-naphthoflavone (BNF). A monoclonal antibody (MAb 1-7-1), which recognizes isozymes of cytochrome P-450 induced by 3-methylcholanthrene (P1-450 and P3-450), selectively inhibited the metabolism of 8-MOP (-57%) and covalent binding of its metabolites (-40%) in microsomes from mice pretreated with BNF, but had no effect in microsomes of mice pretreated with phenobarbital or vehicle. Monoclonal antibody 2-66-3, which recognizes the major isozymes of rat cytochrome P-450 induced by phenobarbital and unknown isozymes in the mouse, enhanced the covalent binding of 8-MOP metabolites in microsomes of mice pretreated with vehicle (+74%), phenobarbital (+44%) or BNF (+31%) without affecting the disappearance of 8-MOP. Preincubation of liver microsomes from BNF-pretreated mice with 40 microM 8-MOP decreased the activity of 7-ethoxycoumarin de-ethylase in a time-dependent manner. Preincubation with 40 microM 8-MOP for 10 min decreased the Vmax from 3.4 to 1.2 nmol/min/mg protein and increased the Michaelis constant from 46 to 90 microM, thus demonstrating mixed competitive and noncompetitive inhibition of 7-ethoxycoumarin de-ethylase. Cysteine trapped three-fourths of the reactive intermediates of 8-MOP but was ineffective in preventing the irreversible inhibition of 7-ethoxycoumarin de-ethylase activity or the 45% spectral loss of cytochrome P-450. Cysteine was ineffective probably because it did not prevent the irreversible binding of metabolites of 8-MOP to cytochrome P-450. There was no spectral evidence that 8-MOP formed cytochrome P-420 or metabolite-intermediate complexes with cytochrome P-450. These findings support the hypothesis that irreversible inactivation of cytochrome P-450 by 8-MOP is caused by modification of the apoprotein by reactive metabolites.
Mays
D C
DC
Department of Pharmacology, College of Medicine, Ohio State University, Columbus.
Hilliard
J B
JB
Wong
D D
DD
Chambers
M A
MA
Park
S S
SS
Gelboin
H V
HV
Gerber
N
N
D015660:7-Alkoxycoumarin O-Dealkylase / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D001571:Benzoflavones / Q000494:pharmacology*; D001665:Binding Sites; D002478:Cells, Cultured; D003545:Cysteine / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology*; D008239:Lysine / Q000494:pharmacology; D008297:Male; D008730:Methoxsalen / Q000378:metabolism*; D051379:Mice; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D010634:Phenobarbital / Q000494:pharmacology; D019324:beta-Naphthoflavone
D000911:Antibodies, Monoclonal; D001571:Benzoflavones; D005419:Flavonoids; D019324:beta-Naphthoflavone; D003577:Cytochrome P-450 Enzyme System; D015660:7-Alkoxycoumarin O-Dealkylase; D008239:Lysine; D003545:Cysteine; D008730:Methoxsalen; D010634:Phenobarbital