Abstract
Cytochromes P450 (CYP) play a major role in drug detoxification, and cytochrome b5 (cyt b5) stimulates the catalytic cycle of mono-oxygenation and detoxification reactions. Collateral reactions of this catalytic cycle can lead to a significant production of toxic reactive oxygen species (ROS). One of the most abundant CYP isoforms in the human liver is CYP2C9, which catalyzes the metabolic degradation of several drugs including nonsteroidal anti-inflammatory drugs. We studied modulation by microsomal membrane-bound and soluble cyt b5 of the hydroxylation of salicylic acid to gentisic acid and ROS release by CYP2C9 activity in human liver microsomes (HLMs) and by CYP2C9 baculosomes. CYP2C9 accounts for nearly 75% of salicylic acid hydroxylation in HLMs at concentrations reached after usual aspirin doses. The anti-cyt b5 antibody SC9513 largely inhibits the rate of salicylic acid hydroxylation by CYP2C9 in HLMs and CYP2C9 baculosomes, increasing the KM approximately threefold. Besides, soluble human recombinant cyt b5 stimulates the Vmax nearly twofold while it decreases nearly threefold the Km value in CYP2C9 baculosomes. Regarding NADPH-dependent ROS production, soluble recombinant cyt b5 is a potent inhibitor both in HLMs and in CYP2C9 baculosomes, with inhibition constants of 1.04 ± 0.25 and 0.53 ± 0.06 µM cyt b5, respectively. This study indicates that variability in cyt b5 might be a major factor underlying interindividual variability in the metabolism of CYP2C9 substrates.
Highlights
Cytochromes P450 (CYP) play a major role in drug detoxification, and cytochrome b5 stimulates the catalytic cycle of mono-oxygenation and detoxification reactions
That cyt b5 significantly inhibits NADPH-dependent H2O2 production in both experimental systems, suggesting that part of the modulatory effect of cyt b5 on CYP2C9 activity might be related to modulation of the production of reactive oxygen species
Our findings show that inhibition of cyt b5 with anti-cyt b5 antibodies sc33174 and sc9513 drastically reduces CYP2C9 activity in two experimental systems, human liver microsomes (HLMs) and baculosomes
Summary
Cytochromes P450 (CYP) play a major role in drug detoxification, and cytochrome b5 (cyt b5) stimulates the catalytic cycle of mono-oxygenation and detoxification reactions. One of the most abundant CYP isoforms in the human liver is CYP2C9, which catalyzes the metabolic degradation of several drugs including nonsteroidal anti-inflammatory drugs. We studied modulation by microsomal membrane-bound and soluble cyt b5 of the hydroxylation of salicylic acid to gentisic acid and ROS release by CYP2C9 activity in human liver microsomes (HLMs) and by CYP2C9 baculosomes. The anti-cyt b5 antibody SC9513 largely inhibits the rate of salicylic acid hydroxylation by CYP2C9 in HLMs and CYP2C9 baculosomes, increasing the KM approximately threefold. Studies in humanized mice have shown that lack of hepatic cyt b5 activity compromises CYP3A4- and CYP2D6-mediated drug metabolism both in vitro and in vivo[12]. Cyt b5 can interact and modulate electron transfer to C YP2C913 probably by interacting with two binding sites in CYP2C9, whose dissociation constants, CYP2C9 / cytochrome P450 reductase, are 0.221 and 0.794 μM cyt b5 for sites 1 and 2, respectively
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