Uncoupling of Cytochrome P450 2B6 and stimulation of reactive oxygen species production in pharmacogenomic alleles affected by interethnic variability

Cytochrome P450 mediated substrate metabolism is generally characterized by the formation of reactive intermediates. In vitro and in vivo reaction uncoupling, results in the accumulation and dissociation of reactive intermediates, leading to increased ROS formation. The susceptibility towards uncoupling and altered metabolic activity is partly modulated by pharmacogenomic alleles resulting in amino acid substitutions. A large variability in the prevalence of these alleles has been demonstrated in CYP2B6, with some being predominantly unique to African populations. The aim of this study is to characterize the uncoupling potential of recombinant CYP2B6*1, CYP2B6*6 and CYP2B6*34 metabolism of specific substrates. Therefore, functional effects of these alterations on Enzyme activity were determined by quantification of bupropion, efavirenz and ketamine biotransformation using HPLC-MS/MS. Determination of H₂O₂ levels was performed by the AmplexRed/ horseradish peroxidase assay. Our studies of the amino acid substitutions Q172H, K262R and R487S revealed an exclusive use of the peroxide shunt for the metabolism of bupropion and ketamine by CYP2B6.K262R. Ketamine was also identified as a trigger for the peroxide shunt in CYP2B6*1 and all variants. Concurrently, ketamine acted as an uncoupler for all enzymes. We further showed that the expressed CYP2B6*34 allele results in the highest H₂O₂ formation. We therefore conclude that the reaction uncoupling and peroxide shunt are directly linked and can be substrate specifically induced with K262R carriers being most likely able to use the peroxide shunt and R487S carrier being most prone to reaction uncoupling. This elucidates the functional diversity of pharmacogenomic in drug metabolism and safety.

CYP2B6; CYP2B6*6; Peroxide shunt; Pharmacogenomics; ROS; Uncoupling.