RNA extraction kits were purchased from Qiagen GmbH (Hilden, Germany). RT-PCR reagents were purchased from Invitrogen (Invitrogen, France). Primers were synthesized by Invitrogen Life Technologies (Invitrogen, France). The LC Fast Start DNA Master SYBR Green I kit was purchased from Roche Diagnostics (Meylan, France), and the Power SYBR Green PCR Master Mix was from Applied Biosystems (Foster City, CA, USA). 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin, 50 μg/mL in DMSO) was obtained from LGC Promochem (Molsheim, France) and Δ⁹-THC (27 mg/mL in ethanol) was from Sigma-Aldrich (Saint Quentin Fallavier, France). Antibodies and supplies used for western blotting were: polyclonal rabbit anti-rat Cyp1a1, polyclonal rabbit anti-Cyp1b1, monoclonal mouse anti-AhR, and monoclonal mouse anti-β-actin antibodies (Abcam, Cambridge, UK and Abcam, Cambridge, MA, USA), horseradish peroxidase-conjugated monkey anti-rabbit secondary antibody and horseradish peroxidase-conjugated monkey anti-mouse secondary antibody (Amersham, Buckinghamshire, UK), and Alexa Fluor^® 488 goat anti-mouse IgG (H+L) antibody (Invitrogen, Carlsbad, CA, USA). Proteins were detected using SuperSignal^® West Pico Chemoluminescent Substrate (Pierce, Rockford, IL, USA). Diesel exhaust particles (DEP) were obtained from NIST 20 (Gaithersburg, MD, USA). Carbon black (CB) mock particles were a kind gift from Degussa Corporation (Akron, OH, USA). AhR antagonist (CH-223191) 21 and IgG control antibody were from Calbiochem-Novabiochem (La Jolla, CA, USA). Protein A/G Beads were from Pierce (Rockford, IL). Other chemicals and reagents were purchased from Sigma-Aldrich (France and St. Louis, MO, USA) or Invitrogen (Cergy-Pontoise, France). Equipments used were: a nucleic acid spectrophotometer (Nanodrop ND-1000, NanoDrop Technologies, USA), a programmable thermal cycler (PTC-100 programmable thermal controller, MJ research Inc., USA), a Light Cycler thermal cycler (Light-Cycler^® instrument, Roche Diagnostics), and a 7900 HT Real-Time PCR Detection System (Applied Biosystems, Foster City, CA).

All animal experiments were based on the same experimental setup. For treatment with TCDD and Δ⁹-THC, male Sprague-Dawley rats (230-250 g) were obtained from Charles River (L'arbresle, France). For treatment with DEP, male CD^® IGS Sprague-Dawley rats (275-300 g) were obtained from Charles River (Portage, MI, USA). Rats were housed in groups of four animals per cage under standard 12:12-hour light/dark conditions (light from 8:00 a.m. to 8 p.m.) in a temperature- and humidity-controlled room. Animals had access to food and water ad libitum. Before using rats for experiments, they were allowed to adapt to the animal facility for 3-5 days.

The care and treatment of animals (TCDD and Δ⁹-THC treatment) were in accordance with standards and guidelines approved by the European Communities Council Directive (86/609/EEC). Animal protocols were approved by the Institutional Animal Care and Use Committees of the EPA, RTP, NC, and the University of Minnesota and were in accordance with AAALAC regulations and the Guides to Animal Use of the University of Minnesota and NIH animal guidelines.

For in vivo TCDD exposure, animals were dosed with a single i.p of TCDD (25 μg/kg body weight) or control (1:9 DMSO: Corn Oil) and sacrificed 72 h after the TCDD or control injection as previously described 22 23 .

For in vivo Δ⁹-THC exposure, Δ⁹-THC (10 mg/kg body weight) or control (1:1:18 ethanol: cremophor: saline) were administered i.p once daily for 7 days. This in vivo dosing protocol was chosen for several reasons: (1) Δ⁹-THC has a half-life of about 25-36 h, which is long enough for a once daily administration, (2) Δ⁹-THC is likely a less potent AhR activator compared to TCDD, (3) the 7-day Δ⁹-THC dosing scheme is currently used in our laboratory, and we observed that it causes an addiction-like behavioral effect (unpublished data). Rats were sacrificed 12 h after the last Δ⁹-THC or control injection.

For in vivo DEP exposure, diesel engine exhaust was generated by operating a 30-kW (40 hp) four-cylinder indirect injection Deutz diesel engine (BF4M1008) and was collected as previously described 24 . Rats in inhalation chambers were exposed to 0.5 mg/m³ and 2 mg/m³ of this diesel engine exhaust for 5 h/day, 5 days/week for 4 consecutive weeks. Inhalation fumes contained diesel exhaust engine gas as well as diesel exhaust particulate matter. These DEP inhalation doses have been used in previous studies and are based on DEP concentrations found in ambient air during heavy traffic 25 26 .

DEP working suspension using diesel exhaust particles (SRM 2975) that were derived from diesel-powered forklifts was prepared as previously described 27 . Briefly, 2 mg of DEP was suspended in 10 mL PBS buffer, vortexed, and sonicated for 30 min at 25°C using an ultrasonic processor (Bransonic^®, Model 3510, Branson Ultrasonic Cooperation, Danbury, CT, USA). The suspension was filtered through a 0.22-μm filter (MillexGS; Millipore, Billerica, MA, USA).

All steps in the isolation of brain microvessels were carried out at 4°C. Rats treated with TCDD, Δ⁹-THC or DEP were anesthetized with isoflurane and sacrificed by decapitation after the last injection or euthanized by CO₂ inhalation and decapitated. Brains were immediately removed and placed in ice-cold HBSS. Cerebella, meninges, brainstems, and large superficial blood vessels were removed. Rat cortex microvessels were isolated according to a protocol that results in the lowest contamination with astrocyte and neuron mRNA and the highest yield of endothelial cells mRNA 28 . Isolated brain microvessels were also free from red blood cells, other contaminating cells such as microglia, and cell debris 27 . Briefly, cortices were minced and homogenized with a Potter-Thomas homogenizer (Konte Glass, Vineland, NJ, USA). The resulting homogenate was centrifuged at 1000 g for 10 min, and the microvessel-enriched pellet was suspended in 17.5% dextran and centrifuged for 15 min at 4400 g at 4°C in a swinging bucket rotor. The resulting pellet was suspended in HBSS containing 1% BSA, while the supernatant containing a layer of myelin was centrifuged once more. The resulting microvessel suspension was passed through a 100 μm nylon mesh, and the filtrate was then passed through a 20 μm nylon mesh. Microvessels retained by the nylon 20 μm mesh were immediately collected and used for experiments or frozen at -80°C.

For ex vivo experiments, freshly isolated brain microvessels from naïve rats were incubated with different concentrations of either TCDD (25 nM) or DEP (0, 5, 50, 200 μg/mL) as previously described 27 .

Total RNA was extracted from tissues and organs of each rat. Total RNA from isolated brain microvessels was obtained by lysing the surrounding basal lamina with proteinase K and then extracting total RNA using the RNeasy Fibrous Tissue Micro kit according to the manufacturer's instructions. RNA samples were purified from contaminating genomic DNA by treatment with DNase (RNase-Free DNase Set, Qiagen SA). RNA concentration and purity were assessed spectrophotometrically at 260 nm using a Nanodrop^® spectrophotometer; RNA integrity was assessed by electrophoresis on a 0.8% agarose gel. One μg of total RNA was reverse transcribed into cDNA in a final volume of 20 μL. The mixture consisted of 1 μg total RNA, 500 μM of each dNTP, 10 mM DTT, 1.5 μM random hexa-nucleotide primers, 20 U RNAse in ribonuclease inhibitor, and 100 U SuperScript II reverse transcriptase. Hexamers were annealed at 25°C for 10 min, products were extended at 42°C for 30 min, and the reaction was terminated by heating to 99°C for 5 min before being quick-chilled to 4°C and stored at -80°C.

RT-PCR for AhR and Cyp1b1 was performed with Taq DNA Polymerase from Promega (Madison, WI, USA) using primers for rat AhR and Cyp1b1 (Table 1) that were custom-synthesized by Qiagen Operon (Alameda, CA, USA). PCR products were resolved on a 2% agarose gel at 100 V for 75 min.

The effect of Δ⁹-THC and TCDD on the expression of Cyp1a1 and Cyp1b1 was investigated by quantitative real-time PCR (q-PCR). ß-actin was used as an endogenous reference for normalizing target gene mRNA. Gene expression was evaluated by q-PCR as previously described 28 . Primers were designed using OLIGO 6.42 software (Medprobe, Norway) (Table 1). PCR reactions were performed on a Light-Cycler^® instrument using the LC-FastStart DNA Master SYBR Green I kit. cDNAs from either control or treated rats were used to generate external calibration standards for each gene. The PCR reaction mixture consisted of 1 μL LC-FastStart DNA Master SYBR Green mix, 1.2 μL of 10 mM MgCl₂, 0.5 μL of each upper and lower primer (final concentration 0.5 μM), and 1.8 μL water. The cDNAs were diluted 40-fold, and 5 μL aliquots were mixed with an equal volume of PCR mixture to yield a final volume of 10 μL. The thermal cycling conditions were 8 min at 95°C followed by 40 amplification cycles at 95°C for 5 s, 64°C for 5 s, and 72°C for 5 s. A target gene was considered to be easily quantifiable when the Ct that was obtained for the less diluted cDNA (1/20) sample was lower than 30. A Ct value of 32 was set as the detection limit. A calibration curve using serial dilutions of cDNA standard was used to determine the relative expression of Cyp1a1 and Cyp1b1 genes in control (vehicle) and TCDD-treated rat (n = 8 rats per group) and in control (vehicle) and Δ⁹-THC-treated rat (n = 12 rats per group). The (fold) change was expressed by the ratio (gene of interest/ß-actin)_treated/(gene of interest/ß-actin)_control.

Freshly isolated brain microvessels were transferred to glass cover slips and fixed for 15 min with 3% paraformaldehyde/0.2% glutaraldehyde at room temperature. After washing with PBS, microvessels were permeabilised for 20 min with 1% (v/v) Triton X-100 in PBS and subsequently blocked with 1% BSA in PBS. Microvessels were incubated overnight at 4°C with the primary antibody to AhR (1:100). After washing with 1% BSA, microvessels were incubated for 1 h at 37°C with Alexa Fluor^® 488-conjugated secondary IgG (1:500, 4 μg/mL; Invitrogen, Eugene, OR, USA); negative controls were incubated with secondary antibody only. Nuclei were counterstained in blue with 2.5 μg/mL DAPI for 15 min. AhR staining was visualised in green using confocal microscopy (Nikon C1 LSC microscope unit, Nikon TE2000 inverted microscope, 40x oil immersion objective, NA 1.3, 488 nm line of an argon laser, 402 nm line of a solid state UV laser; Nikon Instruments Inc., Melville, NY, USA).

Isolated brain microvessels were homogenized in lysis buffer (Sigma, St. Louis, MO, USA) containing Complete^® protease inhibitor (Roche, Mannheim, Germany). The lysate was cleared by centrifugation at 10 000 g for 15 min at 4°C. Identical amounts of pre-cleared microvessel lysates (50 μg of protein) were immunoprecipitated with 5 μg AhR antibody, 5 μg IgG antibody (IgG control), or antibody-free PBS buffer (negative control) by overnight incubation at 4°C. The immune complexes were precipitated with Protein A/G beads (Pierce, Rockford, IL, USA) and washed 3 times with RIPA buffer (Sigma, Dedham, MA, USA) and once with PBS. Immunoprecipitated proteins were eluted with LDS buffer (Invitrogen, Carlsbad, CA, USA) and analyzed by western blotting.

For Cyp1b1, Cyp1a1, and AhR western blots, brain microvessels were homogenized in lysis buffer (Sigma, St. Louis, MO, USA) containing Complete^® protease inhibitor (Roche, Mannheim, Germany) and centrifuged at 10 000 g for 15 min. Denucleated supernatants were used as microvessel lysates, and protein concentrations were determined. Western blots were performed using the Invitrogen NuPage™ Bis-Tris electrophoresis and blotting system (Invitrogen, Carlsbad, CA, USA). After protein transfer, blotting membranes were blocked and incubated with primary antibody (AhR 1:100, Cyp1b1 1:500 (1 μg/mL), Cyp1a1 1:1000, β-actin 1:1000 (1 μg/mL)). Membranes were washed and incubated with horseradish peroxidase-conjugated ImmunoPure^® secondary IgG (1:15,000; Pierce, Rockford, IL, USA) for 1 h. Proteins were detected using SuperSignal^® West Pico Chemoluminescent Substrate (Pierce, Rockford, IL, USA), and bands were visualised and recorded using a BioRad Gel Doc 2000™ gel documentation system (BioRad, Hercules, CA, USA). Rat Cyp1a1 supersomes™ and Cyp1b1 microsomes were supplied by BD Biosciences (Woburn, MA, USA) and used as positive controls for Cyp1a1 and Cyp1b1, respectively.

Statistical analyses were done with GraphPad Prism^® 4.0 software (GraphPad Software Inc., San Diego, CA, USA). Data for qPCR are expressed as means ± SD. Student's unpaired t-tests were performed to identify significant differences between rats treated with TCDD or Δ⁹-THC and control-treated rats. All the tests were two-tailed and statistical significance was set at p < 0.05. Data received from optical density measurements from western blots are means ± SEM.

AhR transcripts (233 bp amplicon) were detected in total brain, brain microvessels, choroid plexus and peripheral tissues (Figure 1A). AhR protein was also detected after immunoprecipitation and western blotting in brain microvessels, which showed a strong signal for AhR (Figure 1B). Importantly, immunoprecipitations without AhR antibody (negative control) or using an IgG control antibody (IgG control) instead of anti-AhR antibody showed no band demonstrating the specificity of the AhR signal. Using immunostaining we localised AhR in the cytoplasm of brain microvessel endothelial cells (Figure 1C), which is consistent with the location of this transcription factor 9 10 . Thus, AhR is expressed at both the mRNA and protein levels in rat brain microvessels.

To demonstrate expression of Cyp1b1, we first performed both RT-PCR and western blotting. High levels of Cyp1b1 transcript (193 bp amplicon, Figure 2A) and protein were detected in brain microvessels, total brain, liver, choroid plexus and kidney (Figure 2B). The apparent molecular weight of Cyp1b1 protein was determined by digital analysis to be 62 kDa, a value that is consistent with the calculated molecular weight of Cyp1b1 at 60.5 kDa.

To test if AhR induces Cyp1a1 and Cyp1b1 genes in isolated brain microvessels, we first incubated ex vivo brain microvessels from naïve rats with 25 nM TCDD, a highly potent AhR ligand, and determined the expression of Cyp1b1 and Cyp1a1, two well-known AhR target genes. In control brain microvessels, Cyp1a1 mRNA was about 60-fold less abundant than that of Cyp1b1. Cyp1b1 and Cyp1a1 mRNA expression levels in brain microvessels were significantly increased by 2.5-fold and 11-fold as early as 3 h after ex vivo TCDD exposure, respectively (Figure 3A).

To test the effect of TCDD on Cyp1a1 and Cyp1b1 in vivo, we dosed animals with TCDD (one dose of 25 μg/kg i.p.) and measured Cyp1a1 and Cyp1b1 transcript levels in isolated brain microvessels with q-PCR at 72 h after TCDD administration. TCDD substantially increased Cyp1b1 and Cyp1a1 mRNA levels in isolated brain microvessels by 22-fold and 260-fold, respectively (Figure 3B). In some vehicle-treated samples, Cyp1a1 transcripts were not detectable and relative expression was therefore calculated using the lowest standard calibration curve.

Δ⁹-THC has been discussed as a potential AhR ligand due to its ability to induce Cyp1a1 expression in a murine hepatoma cell line 17 . Therefore, we studied whether sub-chronic treatment with Δ⁹-THC induces Cyp1b1 and Cyp1a1 in rat brain microvessels. Importantly, Δ⁹-THC did not change Cyp1b1 expression (1.0 ± 0.12 in untreated rats versus 1.1 ± 0.18 in Δ⁹-THC-treated rats, n = 12 per group, data not shown). Cyp1a1 transcripts were not quantified in brain microvessels from control- and Δ⁹-THC-treated rats.

Since TCDD strongly increased mRNA expression of Cyp1b1 and Cyp1a1 in isolated brain microvessels, we measured Cyp1a1 protein levels 72 h after TCDD administration. Cyp1a1 protein was not detected in either TCDD-treated or untreated rats. However, Cyp1a1 was present in rat supersomes™ (positive control) at the correct molecular weight (59 kDa; Figure 4). In contrast, 12 h (Figure 5A) and 72 h (Figure 5B) after TCDD dosing, Cyp1b1 expression was increased by 80 ± 13% and 100 ± 8% (SEM) respectively in TCDD-treated rats compared to control rats.

Since Cyp1a1 was not detected at the protein level after TCDD exposure, we focused on AhR-dependent regulation of Cyp1b1 in the following studies. Diesel exhaust particles (DEP) are a real environmental toxicant that billions of people are exposed to on a daily basis 18 . Once inhaled, DEP can enter the circulation and translocate to tissues throughout the body. Through this route, DEP can reach the brain microvasculature and studies showed that DEP even enter the brain 29 30 31 . DEP consist of a carbon core with adsorbed organic chemicals such as PAHs and HAHs that are known AhR activators 18 19 . Thus, in addition to the DEP itself, the AhR-activating chemicals that are adsorbed to the carbon core also enter the blood-stream and reach the blood-brain barrier where they affect expression of proteins 27 . Here we show that exposing rat brain microvessels to DEP for 6 h ex vivo increased Cyp1b1 protein in a concentration-dependent manner up to 310 ± 16% (SEM) of controls at 200 μg/ml DEP (Figure 6A; data are means from 6 densitometric measurements; brain capillaries used for western blots were pooled from 10 rats). In contrast, carbon black mock particles that are used as a negative control for particulate matter did not cause such an increase in Cyp1b1 protein in brain microvessels (Figure 6B). Note that the difference in band intensities for the controls in Figures 6A and 6B is not due to different expression levels, but to different exposure times. Figure 6C shows that Cyp1b1 protein expression was also significantly increased in brain microvessels from rats that were exposed to 0.5 and 2 mg/m³ diesel engine exhaust containing DEP for 5 h/day, 5 days/week for 4 consecutive weeks (0.5 mg/m³: 447 ± 26%, 2 mg/m³: 538 ± 40% of controls; data are means ± SEM from 5 densitometric measurements; brain capillaries used for western blots were pooled from 6 rats per treatment group). Note that these DEP doses are based on DEP concentrations found in ambient air during heavy traffic 25 26 .

We also addressed the mechanism of DEP-mediated Cyp1b1 induction in rat brain microvessels. Inhibiting transcription with actinomycin D or inhibiting protein synthesis with cycloheximide (CHX) abolished DEP-mediated upregulation of Cyp1b1 protein expression in isolated rat brain microvessels (Figures 7A and 7B). Importantly, the use of the AhR antagonist CH-223191 blocked this DEP-mediated Cyp1b1 induction in brain microvessels (Figure 7C), indicating that this effect is AhR-dependent. Thus, these data demonstrate that DEP induces Cyp1b1 in brain microvessels ex vivo and in vivo and that this effect is likely to be AhR-dependent.