Abstract
Activities of xenobiotic-metabolizing enzymes have been measured with various in vitro and in vivo methods, such as spectrophotometric, fluorometric, mass spectrometric, and radioactivity-based techniques. In fluorescence-based assays, the reaction produces a fluorescent product from a nonfluorescent substrate or vice versa. Fluorescence-based enzyme assays are usually highly sensitive and specific, allowing measurements on small specimens of tissues with low enzyme activities. Fluorescence assays are also amenable to miniaturization of the reaction mixtures and can thus be done in high throughput. 7-Hydroxycoumarin and its derivatives are widely used as fluorophores due to their desirable photophysical properties. They possess a large π-π conjugated system with electron-rich and charge transfer properties. This conjugated structure leads to applications of 7-hydroxycoumarins as fluorescent sensors for biological activities. We describe in this review historical highlights and current use of coumarins and their derivatives in evaluating activities of the major types of xenobiotic-metabolizing enzyme systems. Traditionally, coumarin substrates have been used to measure oxidative activities of cytochrome P450 (CYP) enzymes. For this purpose, profluorescent coumarins are very sensitive, but generally lack selectivity for individual CYP forms. With the aid of molecular modeling, we have recently described several new coumarin-based substrates for measuring activities of CYP and conjugating enzymes with improved selectivity.
Highlights
In a follow-up study [54] we demonstrated, using liquid chromatography (LC)-MS and fluorescence-based assays, that the major metabolic pathway of scoparone to isoscopoletin is 6-O-demethylation in liver microsomes from humans, mice, rats, pigs, and dogs
We developed a simple assay for measuring MFC O-demethylation activity, based on detection of fluorescence emitted by the metabolite 7-hydroxy-4-trifluoromethylcoumarin (HFC)
We identified 3-((3-benzyloxo)phenyl)-7-methoxycoumarin as a new profluorescent substrate of CYP3A4 with higher catalytic rate than with BFC, but it is not selective as it is oxidized by other cytochrome P450 (CYP) forms [41]
Summary
The metabolism of xenobiotics (foreign substances) in a living organism is vitally important to maintain homeostasis. Xenobiotics are transformed in enzyme-catalyzed oxidation, reduction, hydrolysis, and conjugation reactions to more hydrophilic and excretable metabolites. These reactions yield metabolites with diminished biological activity, but sometimes reactive and more toxic intermediates are formed. A cofactor (small endogenous molecule) is transferred to a functional group of a xenobiotic. The functional group is either already present or is created by reactions of oxidation, reduction or hydrolysis. Conjugations are highly important in overall metabolism because they usually inactivate xenobiotics or make reactive metabolites less harmful. Conjugates are often substrates to transporters mediating excretion in the kidney and liver [1,2]
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