Pattern of P450 expression at the human blood-brain barrier: roles of epileptic condition and laminar flow.
OBJECTIVE
P450 enzymes (CYPs) play a major role in hepatic drug metabolism. It is unclear whether these enzymes are functionally expressed by the diseased human blood-brain barrier (BBB) and are involved in local drug metabolism or response. We have evaluated the cerebrovascular CYP expression and function, hypothesizing possible implication in drug-resistant epilepsy.


METHODS
CYP P450 transcript levels were assessed by cDNA microarray in primary endothelial cultures established from a cohort of brain resections (n = 12, drug-resistant epilepsy EPI-EC and aneurism domes ANE-EC). A human brain endothelial cell line (HBMEC) and non-brain endothelial cell line (HUVEC) were used as controls. The effect of exposure to shear stress on CYP expression was evaluated. Results were confirmed by Western blot and immunohistochemistry on brain specimens. Endothelial drug metabolism was assessed by high performance liquid chromatography (HPLC-UV).


RESULTS
cDNA microarray showed the presence of CYP enzymes in isolated human primary brain endothelial cells. Using EPI-EC and HBMEC we found that CYP mRNA levels were significantly affected by exposure to shear stress. CYP3A4 protein was overexpressed in EPI-EC (290 ± 30%) compared to HBMEC and further upregulated by shear stress exposure. CYP3A4 was increased in the vascular compartment at regions of reactive gliosis in the drug-resistant epileptic brain. Metabolism of carbamazepine was significantly elevated in EPI-EC compared to HBMEC.


CONCLUSIONS
These results support the hypothesis of local drug metabolism at the diseased BBB. The direct association between BBB CYP enzymes and the drug-resistant phenotype needs to be further investigated.
Ghosh
Chaitali
C
Department of Cerebrovascular Research, Cleveland Clinic Foundation, Cleveland, Ohio, USA.
Gonzalez-Martinez
Jorge
J
Hossain
Mohammed
M
Cucullo
Luca
L
Fazio
Vincent
V
Janigro
Damir
D
Marchi
Nicola
N
D000704:Analysis of Variance; D000927:Anticonvulsants / Q000494:pharmacology; D001812:Blood-Brain Barrier / Q000187:drug effects / Q000201:enzymology*; D002220:Carbamazepine / Q000378:metabolism; D002461:Cell Line, Transformed; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D042783:Endothelial Cells / Q000201:enzymology; D004730:Endothelium, Vascular; D004827:Epilepsy / Q000201:enzymology / Q000473:pathology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics; D022781:Hepatocytes / Q000166:cytology; D006801:Humans; D012333:RNA, Messenger / Q000378:metabolism; D013314:Stress, Mechanical; D014162:Transfection / Q000379:methods; D014841:von Willebrand Factor / Q000378:metabolism
D000927:Anticonvulsants; D005904:Glial Fibrillary Acidic Protein; D012333:RNA, Messenger; D014841:von Willebrand Factor; D049452:Green Fluorescent Proteins; D002220:Carbamazepine; D003577:Cytochrome P-450 Enzyme System; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human