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Distinction of CYP1A1 and CYP1A2 activity by selective inhibition using fluvoxamine and isosafrole
A Pastrakuljic1, B K Tang, E A Roberts
1Department of Pharmacology, University of Toronto, Ontario, Canada.
Abstract:
Ethoxyresorufin O-deethylation (EROD) has been used as a specific probe for CYP1A1 and CYP1A2. Selective inhibition of one of these cytochromes P450 may differentiate their activity in human liver. Four inhibitors were chosen to examine the selective inhibition of EROD activity, using cDNA of CYP1A1 and CYP1A2. The two flavones, alpha-naphthoflavone and apigenin, while differing in potency, inhibited expressed human CYP1A1, CYP1A2, and human liver microsomes to a similar extent. Isosafrole and fluvoxamine were found to inhibit CYP1A2 selectively, with Ki values of 14 and 800 times, respectively, lower than those for CY1A1. A set of equations was developed to estimate both CYP1A1 and CYP1A2 activity. Levels of CYP1A2 in four human liver specimens ranged from 44.4 to 76.7 pmol/mg protein, which significantly correlated with phenacetin O-deethylase activity (r = 0.99; P < 0.001). Low levels of CYP1A1 activity were present in all four investigated livers, ranging from 0.4 to 2.7 pmol/mg protein.
Insights
Selective inhibitors isosafrole and fluvoxamine can differentiate ethoxyresorufin O-deethylation (EROD) activity between CYP1A1 and CYP1A2 in human liver. This method allows for accurate quantification of these crucial cytochrome P450 enzymes.
Area of Science:
- Pharmacology
- Biochemistry
- Toxicology
Background:
- Ethoxyresorufin O-deethylation (EROD) is a key metabolic pathway.
- CYP1A1 and CYP1A2 are critical cytochrome P450 enzymes involved in xenobiotic metabolism.
- Differentiating CYP1A1 and CYP1A2 activity in human liver is essential for understanding drug metabolism and toxicity.
Purpose of the Study:
- To identify selective inhibitors for differentiating CYP1A1 and CYP1A2 activity.
- To develop a method for quantifying CYP1A1 and CYP1A2 in human liver samples.
- To correlate enzyme activity with specific metabolic markers.
Main Methods:
- Utilized cDNA of CYP1A1 and CYP1A2 to test four inhibitors: alpha-naphthoflavone, apigenin, isosafrole, and fluvoxamine.
- Measured EROD activity and calculated inhibition constants (Ki) for each inhibitor against CYP1A1 and CYP1A2.
- Developed equations to estimate CYP1A1 and CYP1A2 activity in human liver microsomes.
- Assessed CYP1A1 and CYP1A2 levels in human liver specimens and correlated with phenacetin O-deethylase activity.
Main Results:
- Isosafrole and fluvoxamine selectively inhibited CYP1A2 over CYP1A1, with significantly lower Ki values for CYP1A2.
- Alpha-naphthoflavone and apigenin inhibited both CYP1A1 and CYP1A2 similarly.
- Quantified CYP1A2 levels in human livers (44.4–76.7 pmol/mg protein), showing a strong correlation with phenacetin O-deethylase activity (r = 0.99).
- Detected low levels of CYP1A1 activity (0.4–2.7 pmol/mg protein) in all tested human livers.
Conclusions:
- Isosafrole and fluvoxamine are effective selective inhibitors for distinguishing CYP1A2 from CYP1A1 activity in human liver.
- The developed method enables accurate measurement of CYP1A1 and CYP1A2 enzyme activities.
- Findings provide a valuable tool for research in drug metabolism, pharmacokinetics, and toxicology.