Differential Expression of Cytochrome P450 Enzymes from the CYP2C Subfamily in the Human Brain

Abstract

Cytochrome P450 enzymes from the CYP2C subfamily play a prominent role in the metabolic clearance of many drugs. CYP2C enzymes have also been implicated in the metabolism of arachidonic acid to vasoactive epoxyeicosatrienoic acids. CYP2C8, CYP2C9, and CYP2C19 are expressed in the adult liver at significant levels; however, the expression of CYP2C enzymes in extrahepatic tissues such as the brain is less well characterized. Form-specific antibodies to CYP2C9 and CYP2C19 were prepared by affinity purification of antibodies raised to unique peptides. CYP2C9 and CYP2C19 were located in microsomal fractions of all five human brain regions examined, namely the frontal cortex, hippocampus, basal ganglia, amygdala, and cerebellum. Both CYP2C9 and CYP2C19 were detected predominantly within the neuronal soma but with expression extending down axons and dendrites in certain regions. Finally, a comparison of cortex samples from alcoholics and age-matched controls suggested that CYP2C9 expression was increased in alcoholics. IntroductionCytochrome P450 (P450) enzymes play a dominant role in the metabolic clearance of drugs and other environmental chemicals. However, it is increasingly apparent that P450s are expressed differentially in extrahepatic tissues in a region-specific fashion that may influence the tissue-specific clearance of drugs. In particular, P450s are expressed in the brain in a highly cell-specific fashion. Localized metabolism of drugs in the brain may have significant implications for the efficacy and side-effect profile of neuroactive medicines. Given the role of CYP2C forms in the metabolism of a number of drugs affecting the central nervous system (Guengerich, 2005), it is of significant interest to characterize their expression in the brain. Moreover, CYP2C enzymes in animals have been proposed to contribute to the regulation of cerebral blood flow via generation of epoxyeicosatrienoic acid metabolites from arachidonic acid (Alkayed et al., 1996;Iliff et al., 2007). Although a few studies have been undertaken to detect CYP2C transcript expression in the human brain (McFayden et al., 1998;Klose et al., 1999;Dauchy et al., 2008;Dutheil et al., 2009), the detection of CYP2C proteins has been limited by the paucity of human brain tissue and form-specific antibodies. This study aimed to characterize the expression of CYP2C9 and CYP2C19 proteins in discrete regions of the human brain. Materials and MethodsPolyvinylidene fluoride and BioTrace NT nitrocellulose membranes were obtained from Pall Corporation (East Hills, NY). Alexa Fluor 680-and Alexa Fluor 488-labeled goat anti-rabbit IgG antibodies were purchased from Invitrogen (Carlsbad, CA) and IRDye 800-labeled donkey anti-mouse IgG antibody was obtained from Rockland Immunochemicals (Gilbertsville, PA). Mouse antihuman-a-tubulin monoclonal primary antibody was purchased from Sigma (St. Louis, MO). Fluorescence mounting medium (DAKO, Glostrup, Denmark) was used to maintain fluorophore stability.All work described here was done under protocols appro...

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