Abstracts

Rationale: Hepatic nuclear receptors (NRs) and cytochrome P450 enzymes (CYP) are synergistically involved in the metabolism of the majority of drugs. We reported a significant contribution of CYP to drug metabolism by human epileptic blood-brain barrier (BBB) endothelial cells. We expanded these studies to encompass BBB pregnane X (PXR), constitutive androstane (CAR) and glucocorticoid (GR) nuclear receptors, which in the liver are involved in the control of P450 expression. Whether these NRs are responsible for abnormal expression of P450 enzymes in human epileptic brain is unknown.Methods: Surgical brain specimens were obtained from drug resistant epileptic subjects who underwent temporal lobectomies. We used primary cultures of epileptic brain microvascular endothelial cells (EPI-EC, n=8), control brain microvascular endothelial cells (HBMEC, n=8, commercially available) or human hepatocytes (n=3). Nuclear receptor mRNA levels were first screened by cDNA microarrays. The expression of nuclear receptors, PXR, CAR and GR was then quantified by western blotting. CYP and NRs localization in epileptic brain was determined by immunohistochemistry. PXR levels were manipulated by siRNA.Results: Increased mRNA levels of nuclear receptors (NR) were found in epileptic brain endothelial cells compared to control. CYP3A4, 2C9, 2E1 were overexpressed in the majority of EPI-ECs compared to control; in contrast, CYP2D6, 2C19 were down-regulated or absent in EPI-EC. Increased PXR and GR expression was found in EPI-ECs (*p<0.05) compared to HBMEC whereas CAR expression was variable across samples. Hepatocytes consistently showed elevated levels of PXR, CAR and GR (*p<0.01) compared to EPI-ECs and HBMECs. The levels of CYPs positively correlated with PXR and GR, but not to CAR expression in EPI-ECs and controls. CYP-NR expression was localized in the EC, glia and neurons in correspondence with reactive gliosis. The positive correlation between PXR and CYP3A4, CYP2E1 was abolished by silencing of PXR, while CYP2C9 remained unaltered.Conclusions: Our results indicate increase NRs expression in EPI-EC resulting into differential control of selected CYPs. PXR appear to be directly responsible for CYP over-expression at the epileptic cerebrovasculature. Acknowledgements: Supported by R01NS078307 (awarded to NM, DJ and CG). R01NS43284, R41MH093302, R21NS077236, R42MH093302, UH4TR000491, and R21HD057256 awarded to DJ. Brain and Behavior Foundation (NARSAD), American Heart Association (13SDG13950015) and Alternative Research Development Foundation Grant (ADRF) awarded to CG.