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...

Cytochrome P450 enzymes are responsible for the metabolism of most commonly used drugs. Among these enzymes, CYP3A forms mediate the clearance of around 40-50% of drugs and may also play roles in the biotransformation of endogenous compounds. CYP3A forms are expressed both in the liver and extrahepatically. However, little is known about the expression of CYP3A proteins in specific regions of the human brain. In this study, form-selective antibodies raised to CYP3A4 and CYP3A5 were used to characterize the expression of these forms in the human brain. Both CYP3A4 and CYP3A5 immunoreactivity were found to varying extents in the microsomal fractions of cortex, hippocampus, basal ganglia, amygdala, and cerebellum. However, only CYP3A4 expression was observed in the mitochondrial fractions of these brain regions. Nterminal sequencing confirmed the principal antigen detected by the anti-CYP3A4 antibody in cortical microsomes to be CYP3A4. Immunohistochemical analysis revealed that CYP3A4 and CYP3A5 expression was primarily localized in the soma and axonal hillock of neurons and varied according to cell type and cell layer within brain regions. Finally, analysis of the frontal cortex of chronic alcohol abusers revealed elevated expression of CYP3A4 in microsomal but not mitochondrial fractions; CYP3A5 expression was unchanged. The site-specific expression of CYP3A4 and CYP3A5 in the human brain may have implications for the role of these enzymes in both normal brain physiology and the response to drugs.

It is possible that this protein may play a role in the morphological or plastic changes observed after chronic alcohol exposure and withdrawal, either as a cytoskeleton-interacting protein or as a signaling molecule.

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The action of alcohol on neuronal pathways has been an issue of increasing research focus, with numerous findings contradicting the previously accepted idea that its effect is nonspecific. The human NP22 (hNP22) gene was revealed by its elevated expression in the frontal cortex of the human alcoholic. The sequences of hNP22 and the rat orthologue rNP22 contain a number of domains consistent with those of cytoskeletal-interacting proteins. Localization of rNP22 is restricted to the cytoplasm and processes of neurons and it colocalizes with elements of the microfilament and microtubule matrices including filamentous actin (F-actin), alpha-tubulin, tau, and microtubule-associated protein 2 (MAP2). Withdrawal of Wistar rats after alcohol dependence induced by alcohol vapor produced elevated levels of rNP22 mRNA and protein in the cortex, CA2, and dentate gyrus regions of the hippocampus. In contrast, there was decreased rNP22 expression in the striatum after chronic ethanol exposure. Chronic ethanol exposure did not markedly alter rNP22 colocalization with F-actin, alpha-tubulin, or MAP2, although colocalization at the periphery of the neuronal soma with F-actin was observed only after chronic ethanol exposure and withdrawal. Rat NP22 colocalization with MAP2 was reduced during withdrawal, whereas association with alpha-tubulin and actin was maintained. These findings suggest that the effect of chronic ethanol exposure and withdrawal on rNP22 expression is region selective. Rat NP22 may affect microtubule or microfilament function, thereby regulating the neuroplastic changes associated with the development of alcohol dependence and physical withdrawal.