Role of mitochondrial-targeted human cytochrome P450 2D6 in drug metabolism and toxicity

Human cytochrome P450 2D6 (CYP2D6) is responsible for the metabolism of approximately 25% of drugs in common clinical use. The CYP2D6 gene locus is highly polymorphic. Many of the polymorphisms have been shown to be clinically relevant and can account for inter-individual differences in the metabolism of specific drugs. In addition to the established sources of variability in CYP2D6-dependent drug metabolism, we have identified CYP2D6 in the mitochondria of human liver samples and found that extensive inter-individual variability exists with respect to the level of the mitochondrial enzyme. Therefore, in this thesis, I sought to understand the mechanism by which CYP2D6 gets targeted to mitochondria, and whether polymorphic variation has any effect on mitochondrial targeting. I also characterized the metabolic activity of CYP2D6 when it is present in the mitochondria.;In vitro targeting studies demonstrated that CYP2D6 contains an N-terminal chimeric signal that mediates its bimodal targeting to both mitochondria and endoplasmic reticulum. The mitochondrial targeting signal is localized between residues 23-33 and the positively charged residues are required for mitochondrial targeting. Screening of a human liver bank using a reverse genetics approach allowed me to identify several variant forms of CYP2D6 with mutations in the targeting signal region. In vitro targeting studies demonstrated that these mutations can affect the efficiency of mitochondrial targeting.;Most substrates of microsomal CYP2D6 are highly hydrophobic and are able to enter mitochondria, which makes them potential substrates for mitochondrial CYP2D6. My studies demonstrated that human liver mitochondria catalyze the O-demethylation of 7-methoxy-4-aminomethylcoumarin (MAMC), a probe substrate of microsomal CYP2D6, and found that there is significant inter-individual variability in the level of this activity. In addition, mitochondria isolated from a tetracycline-inducible CYP2D6 expression cell line, were found to be active in the metabolism of bufuralol, a classic substrate of microsomal CYP2D6.;The targeting of CYP2D6 to the mitochondria could be a mechanism that protects mitochondria from damage by xenobiotics; however, it could also be a pathologic process that exposes mitochondria to damage by toxic metabolites. The metabolism of 1-methyl-4-pheny1-1,2,3,6-tetrahydropyridine (MPTP) was investigated in order to determine whether mitochondrial CYP2D6 could modulate the mitochondrial toxicity of a xenobiotic substrate. MPTP is a potent neurotoxin that causes significant mitochondrial dysfunction when it gets converted into the toxic metabolite, MPP+. Microsomal CYP2D6 has been implicated in the detoxification of MPTP; however, LC/MS analysis demonstrated that mitochondrial CYP2D6 activates MPTP to form MPP+, and significantly enhances the mitochondrial toxicity of the compound.;Taken together these data suggest that the targeting of CYP2D6 to mitochondria is an important physiological process with a possible role in drug metabolism and drug-induced toxicity.