Aryl hydrocarbon receptor-dependent upregulation of Cyp1b1 by TCDD and diesel exhaust particles in rat brain microvessels

Abstract

Background: AhR activates the transcription of several target genes including CYP1B1. Recently, we showed CYP1B1 as the major cytochrome P450 (CYP) enzyme expressed in human brain microvessels. Here, we studied the effect of AhR activation by environmental pollutants on the expression of Cyp1b1 in rat brain microvessels.

Methods: Expression of AhR and Cyp1b1 was detected in isolated rat brain microvessels. AhR was immunovisualised in brain microvessel endothelial cells. The effect of AhR ligands on Cyp1b1 expression was studied using isolated brain microvessels after ex vivo and/or in vivo exposure to TCDD, heavy hydrocarbons containing diesel exhaust particles (DEP) or Δ9-tetrahydrocannabinol (Δ9-THC).

Results: After ex vivo exposure to TCDD (a highly potent AhR ligand) for 3 h, Cyp1b1 expression was significantly increased by 2.3-fold in brain microvessels. A single i.p. dose of TCDD also increased Cyp1b1 transcripts (22-fold) and Cyp1b1 protein (2-fold) in rat brain microvessels at 72 h after TCDD. Likewise, DEP treatment (in vivo and ex vivo) strongly induced Cyp1b1 protein in brain microvessels. DEP-mediated Cyp1b1 induction was inhibited by actinomycin D, cycloheximide, or by an AhR antagonist. In contrast, a sub-chronic in vivo treatment with Δ9-THC once daily for 7 seven days had no effect on Cyp1b1 expression

Conclusions: Our results show that TCDD and DEP strongly induced Cyp1b1 in rat brain microvessels, likely through AhR activation.

Figure 1

AhR expression in different rat tissues and AhR immunolocalization in isolated rat brain microvessels. (A) RT-PCR of liver, kidney, choroid plexus, total brain, and rat brain microvessels for rat AhR. The RT-PCR shows a signal for AhR mRNA at 233 bp. (B) Western blot of immunoprecipitated AhR from rat brain microvessels. (C) Representative image of a brain microvessel immunostained for AhR (green); nuclei were counterstained with DAPI (blue).

Figure 2

Gene and protein expression of Cyp1b1 in different rat tissues. (A) RT-PCR of Cyp1b1 transcript levels in liver, kidney, choroid plexus, total brain, and isolated brain microvessels. A signal was detected at 193 bp. (B) Western blot showing Cyp1b1 protein expression at 62 kDa.

Figure 3

Effect of TCDD on Cyp1a1 and Cyp1b1 mRNA levels in isolated rat brain microvessels. (A) Freshly isolated rat brain microvessels from naïve rats were incubated ex vivo for 3 h with 25 nM TCDD (n = 4 rats per group). (B) Rats were dosed with a single i.p. dose of 25 μg/kg TCDD and brain microvessels were isolated at 72 h (n = 8 rats per group). Data are shown as relative expression of Cyp1a1 or Cyp1b1 mRNA in treated and control rats using q-PCR. Results are expressed as means ± SD ***p < 0.001 (Student's t-test).

Figure 4

Cyp1a1 protein expression in brain microvessels of TCDD-treated rats. Rats were dosed with a single dose of TCDD (25 μg/kg i.p.) and brain microvessels were isolated after 72 h for Cyp1a1 analysis by Western blotting; rat Cyp1a1 supersomes were used as positive control for Cyp1a1 expression.

Figure 5

Cyp1b1 protein expression in brain microvessels of TCDD-treated rats. Rats were administered a single dose of TCDD (25 μg/kg i.p.), and brain microvessels were isolated 12 h (A) and 72 h (B) after dosing. Rat microsomes were used as positive control for Cyp1b1 expression. The amount of Cyp1b1 in each sample was measured by densitometric analysis and normalized to β-actin.

Figure 6

Effect of DEP exposure on Cyp1b1 expression in isolated rat brain microvessels. Western blots showing Cyp1b1 protein expression; β-actin was used as loading control. (A) Ex vivo DEP exposure of brain microvessels increased Cyp1b1 in a concentration-dependent manner. (B) Carbon black particles were used as a negative control for particulate matter and did not have any effect on Cyp1b1 protein levels in brain microvessels. (C) Cyp1b1 protein expression was increased in brain microvessels from rats that were exposed in vivo to 0.5 and 2 mg/m³ diesel engine exhaust containing DEP for 5 h/day, 5 days/week for 4 consecutive weeks. Brain capillaries were pooled from 6 rats per treatment group.

Figure 7

DEP-mediated Cyp1b1 upregulation involves AhR. Western blots showing Cyp1b1 protein expression; β-actin was used as loading control. DEP-mediated upregulation of Cyp1b1 protein expression in isolated rat brain microvessels is blocked with (A) actinomycin D, an inhibitor of transcription, (B) cycloheximide (CHX), an inhibitor of protein synthesis, and (C) an AhR antagonist (CH-223191).