We have investigated the transcriptomic and/or proteomic patterns of 71 solute carrier (SLC) and organic solute (OST) transporters, 34 ATP-binding cassette (ABC) transporters, and 51 metabolizing enzymes in human brain microvessels. We used quantitative RT-PCR and LC-MS/MS to examine isolated brain microvessels and cortex biopsies from 12 patients with epilepsia or glioma. SLC2A1/GLUT1, SLC1A3/EAAT1, and SLC1A2/EAAT2 were the main SLC proteins whereas ABCG2/BCRP, ABCB1/MDR1, ABCA2 and ABCA8 were the main ABC quantified in isolated brain microvessels; ABCG2/BCRP was 1.6-fold more expressed than ABCB1/MDR1, and ABCC4/MRP4 was 10 times less abundant than ABCB1/MDR1. CYP1B1 and CYP2U1 were the only quantifiable CYPs. Finally, GSTP1, COMT, GSTM3, GSTO1 and GSTM2 proteins were the main phase II enzymes quantified; UGTs and NATs were not detected. Our extensive investigation of gene and protein patterns of transporters and metabolizing enzymes provides new molecular information for understanding drug entry and metabolism in the human blood-brain barrier.

The blood-brain barrier (BBB) is composed of microvessel endothelial cells sealed by tight junctions and surrounded by pericytes, neuron endings and astrocyte foot processes. These form a dynamic neurovascular unit which is the first line of defence for the brain against unwanted compounds. The entry of many compounds into the brain, including numerous commercial drugs, is also restricted by ATPbinding cassette (ABC) efflux transporters, including Pglycoprotein [P-gp, ABCB1/multidrug resistance (MDR1)], several multidrug resistance-associated proteins (MRPs) (ABCCs) and breast cancer resistance protein (BCRP) (ABCG2), at the plasma membrane of brain microvessel Address correspondence and reprint requests to Xavier Declèves, PhD, INSERM U705 CNRS UMR 7157, Faculté de Pharmacie, 4 avenue de l'observatoire, Paris 75006, France. E-mail: xavier.decleves@univ-paris5.frAbbreviations used: ABC, ATP-binding cassette; AhR, aryl hydrocarbon receptor; BBB, blood-brain barrier; BCRP, breast cancer resistance protein; CAR, constitutive androstane receptor; C t , crossing-threshold; CYP, cytochromes P450; GFAP, glial fibrillary acidic protein; MDR, multidrug resistance; MRP, multidrug resistance-associated protein; NG2, neuronglial antigen 2; PECAM-1, platelet endothelial cell adhesion molecule 1; Pgp, P-glycoprotein; PXR, pregnane xenobiotic receptor; qPCR, quantitative PCR; SYP, synaptophysin; TBP, TATA box-binding protein. AbstractWe have established the expression patterns of the genes encoding ATP-binding cassette (ABC) transporters and cytochromes P450 (CYPs) at the adult human blood-brain barrier (BBB) using isolated brain microvessels and cortex biopsies from patients with epilepsia or glioma. Microvessel purity was checked by measuring the expression of genes encoding BBB markers: platelet endothelial cell adhesion molecule 1 (endothelial cells), glial fibrillary acidic protein (astrocytes), synaptophysin (neurons) and neuron-glial antigen 2 (NG2) (pericytes). ABCG2 [breast cancer resistance protein (BCRP)] and ABCB1 (MDR1) were the main ABC transporter genes expressed in microvessels, with 20 times more ABCG2 and 25 times more ABCB1 in microvessels than in the cortex. The CYP1B1 isoform represented over 80% of all the CYPs genes detected in microvessels. There were 14 times more CYP1B1 in microvessels than in the cortex, showing that CYP1B1 is mainly expressed at the BBB. p-glycoprotein (ABCB1), BCRP (ABCG2) and CYP1B1 proteins were found in microvessels by western blotting. The expression of genes encoding three transcription factors [pregnane xenobiotic receptor (PXR), constitutive androstane receptor (CAR), aryl hydrocarbon receptor (AhR)] was also investigated. The AhR gene, involved in the regulation of CYP1B1 expression, was highly expressed in brain microvessels, whereas PXR and CAR genes were almost undetected. This detailed pattern of ABC and CYPs gene expression at the human BBB provides useful information for understanding how their substrates enter the brain.

We investigated the expression of genes encoding ABC transporters, cytochromes P450 (CYPs) and some transcription factors in the hCMEC/D3 immortalized human cerebral microvascular endothelial cell line, a promising in vitro model of the human BBB, and we compared these expressions to a non-brain endothelial cell line (HUVEC) and freshly human brain microvessels. qRT-PCR showed that the MDR1, BCRP, MRP1, MRP3, MRP4 and MRP5 genes were expressed and that the main CYP gene was CYP2U1 in hCMEC/D3. The pattern of ABC and CYPs gene expression in hCMEC/D3 differed from HUVEC which did not express MDR1. Moreover, expression of P-gp and BCRP was lower in hCMEC/D3 than in human brain microvessels but remain functional as shown by rhodamine 123 efflux assay. The gene encoding the aryl hydrocarbon receptor (AhR), a transcription factor that regulates the expression of some ABC and CYPs was highly expressed in hCMEC/D3 and HUVEC, while the pregnane-X-receptor (PXR) and the constitutive androstane receptor (CAR) were barely detected. We investigated the function of the AhR-mediated regulatory pathway in hCMEC/D3 by treating them with the AhR agonist TCDD. The expressions of two AhR-target genes, CYP1A1 and CYP1B1, were increased 26-fold and 28-fold. But the expressions of ABC transporter genes were not significantly altered. We have thus determined the pattern of expression of the genes encoding ABC transporters, CYPs and three transcription factors in hCMEC/D3 and shown that the AhR pathway might afford an original functional transport and metabolic pattern in cerebral endothelial cells that is different from other peripheral endothelial cells.

ABSTRACT:Cytochrome P450 (P450) enzymes and ATP-binding cassette (ABC) transporters modulate the transport and metabolism of both endogenous and exogenous substrates and could play crucial roles in the human brain. In this study, we report the transcript expression profile of seven ABC transporters (ABCB1, ABCC1-C5, and ABCG2), 24 P450s (CYP1, CYP2, and CYP3 families and CYP46A1), and 14 related transcription factors [aryl hydrocarbon receptor, nuclear receptor (NR)1I2/pregnane X receptor, NR1I3/constitutive androstane receptor and NR1C/peroxisome proliferator-activated receptor, NR1H/liver X receptor, NR2B/retinoid X receptor, and NR3A/estrogen receptor subfamilies] in the whole brain, the dura mater, and 17 different encephalic areas. In addition, Western blotting and immunohistochemistry analysis were used to characterize the distribution of the P450s at the cellular and subcellular levels in some brain regions. Our results show the presence of a large variety of xenobiotic transporters and metabolizing enzymes in human brain and show for the first time their apparent selective distribution in different cerebral regions. The most abundant transporters were ABCC5 and ABCG2, which, interestingly, had a higher mRNA expression in the brain compared with that found in the liver. CYP46A1, CYP2J2, CYP2U1, CYP1B1, CYP2E1, and CYP2D6 represented more than 90% of the total P450 and showed selective distribution in different brain regions. Their presence in both microsomal and mitochondrial fractions was shown both in neuronal and glial cells in several brain areas. Thus, our study shows key enzymes of cholesterol and fatty acid metabolism to be present in the human brain and provides novel information of importance for elucidation of enzymes responsible for normal and pathological processes in the human brain.The cytochrome P450 (P450) enzymes belonging to families 1 through 3 and many ATP-binding cassette (ABC) transporters are primarily known for their role in xenobiotic transport and metabolism. These proteins exert their activity mainly in the liver but have also been found in other tissues such as lung, kidney, intestine, placenta, and brain. The extrahepatic localization of these proteins suggests important and unrecognized activities toward endogenous substrates. We hypothesize that the roles of these proteins in transport and metabolism both of endogenous and endogenous compounds are important in the human brain, but at present there is a significant lack of knowledge regarding the distribution of P450s and ABC transporters throughout the human brain.This work was supported by a grant from Servier Technology. Article, publication date, and citation information can be found at

Morphine is well tolerated but addictive, especially for heroin consumers, as heroin is rapidly metabolised to morphine in humans. The hypothesis that psychostimulant drugs, like morphine and/or its withdrawal may cause severe stress that could affect the properties of the blood-brain barrier (BBB) by modifying BBB constitutive markers has been poorly investigated. A recent study has shown that the integrity of the BBB of rats chronically given morphine is altered from 1 day after the last morphine dose, which would be related to the morphine-induced withdrawal syndrome (Sharma and Ali 2006) Abbreviations used: ABC, ATP-binding cassette; BBB, blood-brain barrier; BSA, bovine serum albumin; GFAP, glial fibrillary acidic protein; HIP, hippocampus; Mdr, multidrug resistance; Mrp, multidrug resistance-associated protein; NA, nucleus accumbens; PB, phosphate buffer; PBS, phosphate-buffered saline; P-gp, P-glycoprotein; qRT-PCR, quantitative RT-PCR; RT, room temperature; ZO, zonula occludens. AbstractMorphine may affect the properties of the blood-brain barrier (BBB) by modifying the expression of certain BBB markers. We have determined the effect of chronic morphine treatment on the expression and function of some BBB markers in the rat. The mRNAs of 19 selected genes encoding caveolins, endothelial transporters, receptors and tight junctions proteins in the total RNA of isolated cortex microvessels were assayed by quantitative RT-PCR (qRT-PCR). The expression of genes Mdr1a, Mrp1, Bcrp, Glut-1 and Occludin, was slightly increased, while that of Flk-1 was decreased in microvessels from morphine-treated rats. The expression of the Mrd1a and Mdr1b genes encoding the P-glycoprotein (P-gp) also increased in the whole hippocampus and cortex of morphinetreated rats. The Mdr1a gene induction (1.38-fold) observed by qRT-PCR was also confirmed using in situ hybridization technique (1.40-fold). Immunoblotting revealed an increase in P-gp expression in the hippocampus (1.8-fold) and cortex (1.36-fold) of morphine-treated rats, but no effect in isolated microvessels. In contrast, morphine treatment increased by 1.48-fold the expression of P-gp in a large vessel-enriched fraction. The integrity of the BBB, measured by in situ brain perfusion of [ 14 C]-sucrose, and the activity of P-gp at the BBB, measured with the P-gp substrate [ 3 H]-colchicine, were not modified by morphine. Immunohistofluorescence experiments revealed that P-gp expression is restricted to large vessels and microvessels in control rats and that morphine treatment did not induce the expression of P-gp in the brain parenchyma (astrocytes or neurons). Taken together, our results showed that chronic morphine treatment does not significantly alter BBB integrity or P-gp activity. The impact of morphine-mediated P-gp induction observed in large vessels remains to be determined in terms of brain disposition of drugs that are P-gp substrates.

During the last 2 decades, much evidence concerning the specific distribution patterns of XMET, their induction by xenobiotics and endobiotics and their genetic variations have made cerebral ABC transporters and CYP enzymes key elements in the way individual patients respond to centrally acting drugs.

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