Inhibition of coumarin 7-hydroxylase activity in human liver microsomes

Arch Biochem Biophys. 1997 May 1;341(1):47-61. doi: 10.1006/abbi.1997.9964.


Nine organic solvents and 47 commonly used P450 substrates and inhibitors were examined for their effects on coumarin 7-hydroxylase (CYP2A6) activity in human liver microsomes. Of the nine organic solvents examined (final concentration 1%, v/v), only methanol did not inhibit the 7-hydroxylation of coumarin (0.5 to 50 microM) by human liver microsomes. Dioxane and tetra-hydrofuran, which are structurally related to coumarin, were the most inhibitory solvents examined. Although the rates of coumarin 7-hydroxylation varied enormously among nine samples of human liver microsomes and cDNA-expressed CYP2A6 (Vmax = 179 to 2470 pmol/ mg protein/min), the Km for coumarin 7-hydroxylation was fairly constant (ranging from 0.50 to 0.70 microM). The following chemicals caused little or no inhibition of CYP2A6 as defined by a Ki > 200 microM: caffeine, chlorzoxazone, cimetidine, dextromethorphan, diazepam, diclofenac, erythromycin, ethinylestradiol, ethynyltestosterone, fluconazole, furafylline, furfural, hexobarbital, itraconazole, mephenytoin, methimazole, metronidazole, naringenin, naringin, nifedipine, norfloxacin, norgestrel, orphenadrine, quinidine, papaverine, phenacetin, pyrimethamine, ranitidine, spironolactone, sulfaphenazole, sulfinpyrazone, testosterone, tolbutamide, troleandomycin, and warfarin. In other words, these chemicals, at a final concentration of 100 microM, failed to inhibit CYP2A6 when the concentration of coumarin was equal to Km (0.50 microM). The following chemicals were classified as strong inhibitors of CYP2A6 (defined by Ki < 200 microM): clotrimazole, diethyldithiocarbamate, ellipticine, ketoconazole, 8-methoxypsoralen, 4-methylpyrazole, metyrapone, miconazole, alpha-naphthoflavone, nicotine, p-nitrophenol, and tranylcypromine. The potency with which each chemical inhibited the 7-hydroxylation of coumarin was independent of which sample of human liver microsomes was studied. One of the most potent inhibitors of coumarin 7-hydroxylase was 8-methoxypsoralen (methoxsalen), which was determined to be a mechanism-based inhibitor (suicide substrate) of CYP2A6 (k(inactivation) 0.5 min-1). With the exception of 8-methoxypsoralen, preincubation of human liver microsomes and NADPH with the aforementioned inhibitors did not increase their ability to inhibit CYP2A6. The most potent competitive inhibitor of CYP2A6 was tranylcypromine (Ki = 0.04 microM). Several of the chemicals that strongly inhibited CYP2A6, such as ketoconazole and tranylcypromine, are often used with the intention of selectively inhibiting human P450 enzymes other than CYP2A6. The results of this study underscore the need for a systematic evaluation of the specificity of commonly used P450 inhibitors.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adult
  • Aryl Hydrocarbon Hydroxylases*
  • Child, Preschool
  • Coumarins / metabolism
  • Cytochrome P-450 CYP2A6
  • Cytochrome P-450 Enzyme Inhibitors*
  • Cytochrome P-450 Enzyme System / metabolism
  • Dioxanes / pharmacology
  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / pharmacology*
  • Female
  • Furans / pharmacology
  • Humans
  • Hydroxylation / drug effects
  • Kinetics
  • Male
  • Microsomes, Liver / enzymology*
  • Middle Aged
  • Mixed Function Oxygenases / antagonists & inhibitors*
  • Mixed Function Oxygenases / metabolism
  • Solvents / pharmacology*
  • Umbelliferones / metabolism


  • Coumarins
  • Cytochrome P-450 Enzyme Inhibitors
  • Dioxanes
  • Enzyme Inhibitors
  • Furans
  • Solvents
  • Umbelliferones
  • tetrahydrofuran
  • 7-hydroxycoumarin
  • Cytochrome P-450 Enzyme System
  • coumarin
  • Mixed Function Oxygenases
  • Aryl Hydrocarbon Hydroxylases
  • CYP2A6 protein, human
  • Cytochrome P-450 CYP2A6
  • 1,4-dioxane