Xavier Declèves

ABC transporters, cytochromes P450 and their main transcription factors: expression at the human blood–brain barrier

We 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. Microves 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.

Transcriptomic and Quantitative Proteomic Analysis of Transporters and Drug Metabolizing Enzymes in Freshly Isolated Human Brain Microvessels

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.

Functional expression of P-glycoprotein and multidrug resistance-associated protein (Mrp1) in primary cultures of rat astrocytes.

Although it has been well established that the drug efflux pump P‐glycoprotein (P‐gp) protects the brain against the entry of cytotoxic drugs, its real in situ localization, i.e., at brain capillary endothelial cells or on astrocyte foot processes, is still controversial. The aim of this study was to compare the expression of P‐gp and of multidrug resistance‐associated protein (Mrp1), another drug efflux pump, in cultured neonatal rat brain astrocytes and in cultured brain capillary endothelial cells. Reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis showed that the mdr1b gene was preferentially expressed in astrocytes, whereas both mdr1a and mdr1b mRNA were detected in endothelial cells. Moreover, the mrp1 gene encoding Mrp1 was expressed in both cell types. Western blotting analysis revealed higher expression of P‐gp in endothelial cells as compared with astrocytes, but higher expression of Mrp1 in astrocytes. Moreover, P‐gp and Mrp1 expression was not modified in more differentiated astrocytes obtained when cultured with db‐cAMP for 48 hr. Our functional analysis of P‐gp showed a modest effect of P‐gp modulators (CsA, verapamil, PSC 833) on the uptake of colchicine (a substrate of P‐gp) by astrocytes, whereas they increased by about 50% the uptake of vincristine (a common substrate of P‐gp and MRP) by astrocytes. MRP modulators (genistein, probenecid, and sulfinpyrazone) did not modify the uptake of colchicine but increased that of vincristine with a major effect found for sulfinpyrazone. Moreover, indomethacin, probenecid, and sulfinpyrazone increased the uptake of fluorescein (a substrate of MRP but not of P‐gp). Taken together, our results provide the first biochemical and functional evidence supporting the expression of P‐gp and Mrp1 in rat cultured astrocytes. J. Neurosci. Res. 60:594–601, 2000

Expression and transcriptional regulation of ABC transporters and cytochromes P450 in hCMEC/D3 human cerebral microvascular endothelial cells

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.

Xenobiotic-metabolizing enzymes and transporters in the normal human brain: regional and cellular mapping as a basis for putative roles in cerebral function.

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.

A functional in vitro model of rat blood-brain barrier for molecular analysis of efflux transporters

Physiological studies of the blood-brain barrier (BBB) are often performed in rats. We describe the functional characterization of a reproducible in vitro model of the rat BBB and its validation for investigating mechanisms involved in BBB regulation. Puromycin-purified primary cultures of brain endothelial cells, co-cultured with astrocytes in the presence of hydrocortisone (HC) and cAMP, presented low sucrose permeability (< or =0.1 x 10(-3) cm/min) and high transendothelial electrical resistance (> or =270 Omega cm(2)). Expression of specific BBB markers and their transcripts was detected by immunostaining and RT-PCR, respectively: tight junction proteins (claudin-3 and -5, ZO-1 and occludin) and transporters (P-gp, Bcrp and Oatp-2). RT-PCR experiments demonstrated a role of treatment by astrocytes, HC and cAMP in regulation of the transcript level of tight junction proteins (claudin-5 and ZO-1) as well as transporters (Mdr1a, Mrp3, Mrp4, Bcrp, Glut-1), while transcript level of Mdr1b was significantly decreased. The functionality of efflux pumps (P-gp, Mrps and Bcrp) was demonstrated in the presence of specific inhibitors (PSC833, MK571 or Ko143, respectively) by (i) assessing the uptake of the common substrates rhodamine 123 and daunorubicin and (ii) evaluating apical to basolateral and basolateral to apical polarized transport of daunorubicin. In addition, a good correlation (R=0.94) was obtained between the permeability coefficients of a series of compounds of various lipophilicity and their corresponding in vivo rodent blood-brain transfer coefficients. Taken together, our results provide compelling evidence that puromycin-purified rat brain endothelial cells constitute a reliable model of the rat BBB for physiological and pharmacological characterization of BBB transporters.

Expression of drug transporters at the blood–brain barrier using an optimized isolated rat brain microvessel strategy

Quantitative RT-PCR (qRT-PCR) and Western blotting studies on transporters at the blood-brain barrier (BBB) of isolated brain microvessels have produced conflicting data on their cellular distribution. A major problem is identifying cells expressing the genes of interest, since isolated brain microvessels are composed of several cell types and may be contaminated with mRNA or proteins from astrocytes and neurons. We isolated rat brain microvessels and examined microscopically samples at each step of isolation to evaluate microvessel purity. The expression of specific markers of endothelial cells (Glut-1, Flk-1), pericytes (Ng2), neurons (synaptophysin, Syn) and astrocytes (Gfap) was measured by qRT-PCR in order to select the protocol giving the least astrocyte and neuron mRNAs and the most endothelial mRNAs. We also evaluated the gene expression of drug transporters (Mdr1a, Mdr1b, Mrp1-5, Bcrp and Oatp-2) at each step to optimize their location in cells at the BBB. The Mdr1a, Mrp4, Bcrp and Oatp-2 gene profiles were similar to those of endothelium markers. The profiles of Mrp2 and Mrp3 closely resembled that of Ng2. Mrp5 and Mrp1 expression was not increased in the microvessel-enriched fraction, suggesting that they are ubiquitously expressed throughout the cortex parenchyma. We also evaluated by Western blotting the expression of P-gp, Mrp2, Gfap and Syn in the cortex and in the purest obtained microvessel fraction. Our results showed that P-gp expression strongly increased in microvessels whereas Mrp2 was not detected in any of the fraction. Surprisingly, Gfap expression increased in isolated microvessels whereas Syn was not detected. Our results showed that the strategy consisting of identifying gene expression at different steps of the protocol is useful to identify cells containing mRNA at the BBB and give overall similar results with protein expression.

Quantitative targeted absolute proteomic analysis of transporters, receptors and junction proteins for validation of human cerebral microvascular endothelial cell line hCMEC/D3 as a human blood-brain barrier model.

Human cerebral microvascular endothelial cell line hCMEC/D3 is an established model of the human blood-brain barrier (BBB). The purpose of the present study was to determine, by means of quantitative targeted absolute proteomics, the protein expression levels in hCMEC/D3 cells of multiple transporters, receptors and junction proteins for comparison with our previously reported findings in isolated human brain microvessels. Among 91 target molecules, 12 transporters, 2 receptors, 1 junction protein and 1 membrane marker were present at quantifiable levels in plasma membrane fraction of hCMEC/D3 cells. ABCA2, MDR1, MRP4, BCRP, GLUT1, 4F2hc, MCT1, ENT1, transferrin and insulin receptors and claudin-5 were detected in both hCMEC/D3 cells and human brain microvessels. After normalization based on Na(+)/K(+) ATPase expression, the differences in protein expression levels between hCMEC/D3 cells and human brain microvessels were within 4-fold for these proteins, with the exceptions of ENT1, transferrin receptor and claudin-5. ABCA8, LAT1, LRP1 and γ-GTP were below the limit of quantification in the cells, but were found in human brain microvessels. ABCA3, ABCA6, MRP1 and ATA1 were found only in hCMEC/D3 cells. Furthermore, compared with human umbilical vein endothelial cells (HUVECs) as reference nonbrain endothelial cells, MDR1 was found only in hCMEC/D3 cells, and GLUT1 expression was 15-fold higher in hCMEC/D3 cells than in HUVECs. In conclusion, this is the first study to examine the suitability and limitations of the hCMEC/D3 cell line as a BBB functional model in terms of quantitative expression levels of transporters, receptors and tight junction proteins.

Molecular and functional MDR1-Pgp and MRPs expression in human glioblastoma multiforme cell lines.

The aim of our study was to investigate the functional expression of P‐glycoprotein (Pgp) and multidrug resistance‐associated proteins (MRPs) in 2 distinct glioma cells (GL15 and 8MG) from patients with glioblastoma multiforme. MDR1 gene and Pgp expression was not detected in either cell line by RT‐PCR and Western blotting, respectively. In contrast, MRP1 was detected at both mRNA and protein level in both cell lines, with a higher expression in the 8MG cells that occur predominantly at the cell membrane. Three other MRPs (MRP3, MRP4 and MRP5) were detected by RT‐PCR in both cell lines, whereas MRP2 was not expressed. In addition, MRP3 protein was also detected by immunocytochemistry in both GL15 and 8MG cell lines. Indomethacin and probenecid, 2 modulators of MRPs activity, increased the accumulation of vincristine and etoposide, 2 substrates of MRPs, by both cell lines. These modulators also decreased the efflux of vincristine from both cell lines with a more pronounced effect in 8MG cells. In conclusion, our results show functional expression of MRPs leading to a decrease in the intracellular vincristine and etoposide concentrations in human glioblastoma cell lines. Furthermore, our results that exhibit protein expression of MRP1 and MRP3 and gene expression of MRP4 and MRP5 in these 2 glioblastoma cell lines suggest new mechanisms that could lead to a MDR phenotype of tumour cells in patients with glioblastoma multiforme.

Interaction of drugs of abuse and maintenance treatments with human P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2)

Drug interaction with P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) may influence its tissue disposition including blood-brain barrier transport and result in potent drug-drug interactions. The limited data obtained using in-vitro models indicate that methadone, buprenorphine, and cannabinoids may interact with human P-gp; but almost nothing is known about drugs of abuse and BCRP. We used in vitro P-gp and BCRP inhibition flow cytometric assays with hMDR1- and hBCRP-transfected HEK293 cells to test 14 compounds or metabolites frequently involved in addiction, including buprenorphine, norbuprenorphine, methadone, ibogaine, cocaine, cocaethylene, amphetamine, N-methyl-3,4-methylenedioxyamphetamine, 3,4-methylenedioxyamphetamine, nicotine, ketamine, Delta9-tetrahydrocannabinol (THC), naloxone, and morphine. Drugs that in vitro inhibited P-gp or BCRP were tested in hMDR1- and hBCRP-MDCKII bidirectional transport studies. Human P-gp was significantly inhibited in a concentration-dependent manner by norbuprenorphine>buprenorphine>methadone>ibogaine and THC. Similarly, BCRP was inhibited by buprenorphine>norbuprenorphine>ibogaine and THC. None of the other tested compounds inhibited either transporter, even at high concentration (100 microm). Norbuprenorphine (transport efflux ratio approoximately 11) and methadone (transport efflux ratio approoximately 1.9) transport was P-gp-mediated; however, with no significant stereo-selectivity regarding methadone enantiomers. BCRP did not transport any of the tested compounds. However, the clinical significance of the interaction of norbuprenorphine with P-gp remains to be evaluated.