Cellular localization and functional significance of CYP3A4 in the human epileptic brain

Abstract

SUMMARYPurpose: Compelling evidence supports the presence of P450 enzymes (CYPs) in the central nervous system (CNS). However, little information is available on the localization and function of CYPs in the drug-resistant epileptic brain. We have evaluated the pattern of expression of the specific enzyme CYP3A4 and studied its co-localization with MDR1. We also determined whether an association exists between CYP3A4 expression and cell survival. Methods: Brain specimens were obtained from eight patients undergoing resection to relieve drug-resistant seizures or to remove a cavernous angioma. Each specimen was partitioned for either immunostaining or primary culture of human endothelial cells and astrocytes. Immunostaining was performed using anti-CYP3A4, MDR1, GFAP, or NeuN antibodies. High performance liquid chromatography-ultraviolet (HPLC-UV) analysis was used to quantify carbamazepine (CBZ) metabolism by these cells.CYP3A4 expression was correlated to DAPI) condensation, a marker of cell viability. Human embryonic kidney (HEK) cells were transfected with 4¢,6-diamidino-2-phenylindole (CYP3A4 to further evaluate the link between CYP3A4 levels, CBZ metabolism, and cell viability. Key Findings: CYP3A4 was expressed by blood-brain barrier (BBB) endothelial cells and by the majority of neurons (75 ± 10%). Fluorescent immunostaining showed coexpression of CYP3A4 and MDR1 in endothelial cells and neurons. CYP3A4 expression inversely correlated with DAPI nuclear condensation. CYP3A4 overexpression in HEK cells conferred resistance to cytotoxic levels of carbamazepine. CYP3A4 levels positively correlated with the amount of CBZ metabolized. Significance: CYP3A4 brain expression is not only associated with drug metabolism but may also represent a cytoprotective mechanism. Coexpression of CYP3A4 and MDR1 may be involved in cell survival in the diseased brain.

Blood-Brain Barrier P450 Enzymes and Multidrug Transporters in Drug Resistance: A Synergistic Role in Neurological Diseases

“…Moreover, the possibility exists that brain CYPs may be mutated and operate differently compared to the hepatic ones [23,24]. The latter represents an interesting, yet unstudied, hypothesis.…”

The Blood-Brain Barrier in Epilepsy

“…Neuronal CYP3A4 expression has also been demonstrated in brain sections by immunostaining from patients with temporal lobe epilepsy, tuberous sclerosis, or cavernous angioma, all of whom had intractable epilepsy. In these samples, CYP3A4 was rarely co-localised with the astrocytic marker GFAP (Ghosh et al, 2011). Carbamazepine given to cells that derived from resected epileptic brain tissue underwent metabolism at an extent similar to what was observed in hepatocytes.…”

“…But carbamazepine is metabolized by CYP3A4 (, Table 1), and high expression of CYP3A4 protein was found in endothelial cells isolated from surgically resected epileptic brain tissue (Ghosh et al, 2011). Neuronal CYP3A4 expression has also been demonstrated in brain sections by immunostaining from patients with temporal lobe epilepsy, tuberous sclerosis, or cavernous angioma, all of whom had intractable epilepsy.…”

“…27 gamma-glutamyl transpeptidase (Beuckmann et al, 1995), alkaline phosphatase (Beuckmann et al, 1995), aromatic L-amino acid decarboxylase (Betz et al, 1980;Matter & Balda, 2003b), the phase I metabolising enzymes CYP1A1 (Filbrandt et al, 2004), CYP1B1 (Filbrandt et al, 2004), CYP3A4 (Ghosh et al, 2010;Ghosh et al, 2011), andCyp4x1 (AlAnizy et al, 2006), NADPH-CYP P450 reductase (Chat et al, 1998;Ghersi-Egea et al, 1988;Minn et al, 1991;Ravindranath et al, 1990), epoxide hydrolase (Ghersi-Egea et al, 1988;Minn et al, 1991), and the phase II enzymes, 1-naphthol-UDP-glucuronosyltransferase (Ghersi-Egea et al, 1988) and GSTμ (Shang et al, 2008), and GSTπ (Bauer et al, 2008;Shang et al, 2008). The presence of these enzymes in the brain microvasculature indicates the existence of a metabolic barrier.…”

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].…”