Dealkylation of 7-methoxycoumarin as assay for measuring constitutive and phenobarbital-inducible cytochrome P450s.
Six alkyl ethers of 7-hydroxycoumarin, ranging from methoxy- to hexoxycoumarin, were studied for their NADPH-dependent metabolism by liver microsomes of male rats treated with phenobarbital (PB) or 3-methyl-cholanthrene (MC). The six alkyl ethers were metabolized by both types of microsomes, forming 7-hydroxycoumarin as the major product. Among the test compounds, 7-methoxycoumarin was unusual in that its dealkylation was inducible only by PB and not by MC. PB increased 7-methoxycoumarin-O-demethylase (MOCD) activity about four- to eightfold. Metyrapone strongly inhibited MOCD in PB-treated microsomes but not in MC-treated microsomes. Similarly, monoclonal antibodies directed toward PB-induced cytochrome P450s selectively suppressed MOCD in PB-treated microsomes. MOCD activity was observed in preparations of SD1 cells containing only cytochrome P450IIB1, while it was not found in preparations of XEM1 cells containing only cytochrome P450IA1. Demethylation of 7-methoxycoumarin was also mediated by the constitutive cytochrome P450 form(s) of liver, lung, small intestine, and kidney (in decreasing order). PB increased MOCD activity of small intestine by 40% but was without effect on the dealkylation activity of lung and kidney. MOCD activity was also detectable in differentiated rat hepatoma lines H4IIEC3 and 2sFou. The studies indicate that dealkylation of 7-methoxycoumarin is a highly sensitive, simple, and practical assay for estimating constitutive and PB-inducible cytochrome P450-dependent monooxygenase activities.
Reen
R K
RK
Biochemistry Section, Regional Research Laboratory, Jammu, India.
Ramakanth
S
S
Wiebel
F J
FJ
Jain
M P
MP
Singh
J
J
D015660:7-Alkoxycoumarin O-Dealkylase / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002460:Cell Line; D006224:Cricetinae; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000378:metabolism*; D003640:Dealkylation; D004790:Enzyme Induction; D006863:Hydrogen-Ion Concentration; D008114:Liver Neoplasms, Experimental; D008297:Male; D008722:Methods; D008862:Microsomes, Liver / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010634:Phenobarbital / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D014468:Umbelliferones / Q000737:chemistry / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D014468:Umbelliferones; D009249:NADP; C031477:7-hydroxycoumarin; D003577:Cytochrome P-450 Enzyme System; D015660:7-Alkoxycoumarin O-Dealkylase; D010634:Phenobarbital