@article {Mays720, author = {D C Mays and J B Hilliard and D D Wong and M A Chambers and S S Park and H V Gelboin and N Gerber}, title = {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.}, volume = {254}, number = {2}, pages = {720--731}, year = {1990}, publisher = {American Society for Pharmacology and Experimental Therapeutics}, abstract = {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.}, issn = {0022-3565}, URL = {https://jpet.aspetjournals.org/content/254/2/720}, eprint = {https://jpet.aspetjournals.org/content/254/2/720.full.pdf}, journal = {Journal of Pharmacology and Experimental Therapeutics} }