Differential Expression of Human Cytochrome P450 Enzymes from the CYP3A Subfamily in the Brains of Alcoholic Subjects and Drug-Free Controls

Abstract

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.

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

“…Prior to injection of antigen, 20 ml of preimmune serum was collected from each animal to check background immunogenicity. Animals were exsanguinated 11 weeks after initial immunization, whole blood was collected, IgG fractions were prepared, and antibodies were affinity purified as previously described (Booth Depaz et al, 2013) using bacterial membrane preparations containing recombinant CYP2C enzymes. Isolated antibodies raised against CYP2C9, CYP2C19, or CYP2C18 were tested for specificity to all four recombinant human CYP2C proteins (CYP2C9, CYP2C19, CYP2C8, and CYP2C18) expressed in bacterial membrane fractions (Supplemental Table 1) (Cuttle et al, 2000;Shukla et al, 2005).…”

“…Samples used to compare the expression of P450s in the alcoholic and nonalcoholic brain were obtained from 12 different male subjects matched for age (Supplemental Table 3). Brain microsomes were prepared as previously described (Booth Depaz et al, 2013) from approximately 500 mg frozen brain tissue.…”

“…Either 10 mg (CYP2C18 antibody) or 20 mg protein (CYP2C9 and CYP2C19 antibodies) was subjected to immunoblotting as previously described (Booth Depaz et al, 2013). b-actin immunoreactivity detected with a mouse anti2b-actin monoclonal primary antibody (used at a dilution of 1:20,000; Sigma) was used as a loading control.…”

“…Data were subjected to t test analysis with a 95% confidence interval. Fluorescent immunohistochemistry of paraffin-embedded sections was done as previously described using an Alexa Fluor 488-labeled goat anti-rabbit IgG secondary antibody (Booth Depaz et al, 2013).…”

CYP-13A12 of the nematode Caenorhabditis elegans is a PUFA-epoxygenase involved in behavioural response to reoxygenation

“…C. elegans harbours several CYP genes that are homologous to mammalian CYP isoforms [11]. CYP3A4, a CYP enzyme predominantly expressed in the liver, but also in the brain and other extrahepatic tissues [12,13], is the most closely related human homolog of CYP-13A12 (32 % amino acid identity). Human CYP3A4 has been primarily known for its important role in liver microsomal drug metabolism but also contributes to the metabolism of a wide variety of endogenous substrates including the epoxidation of AA (arachidonic acid; C 20:4, n−6 ) and anandamide [14,15].…”