Expression of drug metabolizing enzymes in choroid plexus. Implications in detoxification processes



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FARMACIA, 2008, Vol.LVI, 3

EXPRESSION OF DRUG METABOLIZING ENZYMES IN CHOROID PLEXUS. IMPLICATIONS IN DETOXIFICATION PROCESSES
DANIELA GRĂDINARU1*, ANNE-LAURE MINN2, JEAN-MARIE HEYDEL2, YVES ARTUR2, NICULINA MITREA1*

1Department of Biochemistry, Faculty of Pharmacy, University of Medicine and Pharmacy “Carol Davila”, Bucharest, Romania

2Unité de Biochimie-Pharmacologie, Faculté de Medicine et de Pharmacie, Université de Bourgogne, 21033 Dijon, France

*corresponding author: biochimiebucuresti@yahoo.com
Abstract

The choroid plexus (CP), which is the principal site of formation of the cerebrospinal fluid (CSF), controls the exchange of many endogenous compounds and exogenous molecules between brain tissue and CSF. We present the changes in mRNA expression and enzymatic activities of UDP-glucuronosyltransferase UGT1A6 isoform and NADPH-cytochrome P450 reductase, after “in vitro” treatment with xenobiotic molecules known to act in the liver as inducers or inhibitors of these drug metabolizing enzymes. Five study groups of male Sprague-Dawley rats were treated separately with: 3-methylcholantrene (3-MC), phenobarbital (PB), dexamethasone (DX), cyclosporine (CY) or paraquat (PQ). Choroidal 1-naphthol glucuronidation activities were significantly induced by 3-MC and PQ administration (354±85 and 257±49 vs. 115±24 nmoles/hour/mg protein, in control group) whereas the other molecules were without effect. UGT1A6 mRNA expression, measured by reverse transcription- polymerase chain reaction (RT-PCR), was 2.3 fold higher after 3-MC treatment and 2.1 fold higher after PQ administration. By contrast, reductasic activities and mRNA expression remained unchanged in the isolated CP, in these experimental conditions. We present for the first time evidences that the CP express these both phase I and II drug metabolizing-enzymes, and that mRNA expression can be regulated by exogenous factors. These results emphasize the role of CP in the metabolic protection to the brain against xenobiotics.
Rezumat

Plexurile coroide (PC) reprezintă sediul principal al formării lichidului cefalo-rahidian (LCR) controlând în acelaşi timp schimbul a numeroşi compuşi endogeni şi exogeni intre ţesutul cerebral şi LCR. Sunt prezentate schimbările în exprimarea la nivelul ARNm şi în activităţile enzimatice ale UDP-glucuroniltransferazei- izoforma UGT1A6, şi cele ale NADPH-citocrom P450 reductazei în urma tratamentului „in vitro” cu diferite molecule xenobiotice cunoscute pentru efectul lor inductor sau inhibitor, la nivel hepatic, asupra acestor enzime de metabolizare a medicamentelor. Şobolani masculi Sprague-Dawley împârţiţi în cinci loturi de studiu au fost trataţi separat cu: 3-metilcolantren (3-MC), fenobarbital (PB), dexametazonă (DX), ciclosporină (CY) sau paraquat (PQ). Activităţile de glucuronoconjugare pentru 1-naftol la nivel coroidal au fost semnificativ induse în urma administrării 3-MC şi PQ (354±85 şi 257±49 faţă de 115±24 nmoli/oră/mg proteină, pentru grupul de control), în timp ce restul moleculelor nu au avut nici un efect. Expresia UGT1A6 la nivelul ARNm, determinată prin transcriere inversă şi reacţie de polimerizare în lanţ (RT-PCR), a fost de 2.3 ori mai mare în urma tratamentului cu 3-MC şi de 2.1 ori mai mare în urma administrării PQ. Spre deosebire de acestea, activităţile şi expresia ARNm pentru reductază au rămas neschimbate. Prezentăm pentru prima dată dovezi privind exprimarea acestor enzime de metabolizare a medicamentelor de fază I şi II in plexurile coroide, şi reglarea acestei expresii de către factori exogeni. Aceste rezultate subliniză rolul plexurilor coroide în protecţia metabolică a creierului împotriva acţiunii compuşilor xenobiotici.

  • choroids plexus

  • drug metabolizing enzymes

  • glucuronidation


INTRODUCTION

The choroid plexus (plexus choroidei), located in the lateral, third and fourth ventricles, is the site of production of the cerebrospinal fluid (CSF). Active efflux transport systems, as well as metabolic enzymes in the choroid plexus epithelial cells, which form a tight monolayer, play a protective role by facilitating the elimination of xenobiotics including drugs and endogenous waste from the CSF to prevent their accumulation in the central nervous system [1]. Therefore, choroids plexus forms most of the blood-CSF barrier, which efficiently maintains CSF chemical stability [2].

Moreover, convincing evidence has been presented that some xenobiotics undergo biotransformation during this exchange [3]. Thus the precise knowledge of the enzymes involved in this metabolic pathway may allow to use the choroids plexus as a tool to administrate some drugs that do not cross the blood-brain barrier (BBB). Previous work demonstrated that isolated rat choroids plexus displays a very high, age-dependent and 3-methylcholanthrene inducible glucuronidation enzymatic activity towards potentially neurotoxic xenobiotics, 1-naphthol and 4-methyl-umbelliferone [4]. These results suggested that the choroids plexus, like the BBB, affords a metabolic protection to the brain against xenobiotics [5].

Glucuronidation represents a major detoxification pathway, catalyzed by UDP-glucuronosyltransferases (UGTs, EC: 2.4.1.17) which belong to a multigenic family of enzymes that are mainly expressed in the liver endoplasmic reticulum, but conjugation activities of several isoforms have been described in the cerebral and olfactory tissues [6,7,8].

NADPH-cytochrome P450 reductase (EC: 1.6.2.4) is a microsomal flavoprotein which supply reducing equivalents from NADPH to cytochromes P450 (CYP) during its catalytic cycle, thereby enabling the heme protein to activate molecular oxygen and to metabolize a variety of endogenous and exogenous lipophilic compounds. This oxidative enzyme displays a high activity in isolated brain microvessels and choroids plexus [9].

The purpose of this study was to determine whether UGT1A6 and NADPH-cytochrome P450 reductase expression and enzymatic activities are regulated by drug metabolizing enzymes inducers known to act as ligands or activators for the aryl hydrocarbon receptor (AhR): 3-methylcholanthrene, the constitutive androstane receptor (CAR): phenobarbital, or the pregnane X receptor (PXR): dexamethasone. Cyclosporine and paraquat were used to evaluate whether the reactive oxygen species generation mediated by their metabolism could modulate the expression of these choroidal enzymes.


MATERIALS AND METHODS

Animal experiments

Male Sprague-Dawley (250±15g, Iffa-Credo, St Germain–sur-l’Arbresle, France) rats aged 3 months, were maintained with a 12 h/12 h light/dark cycle and received standard laboratory food and water “ad libitum”. In order to evaluate the effects of potential inducers/repressors on enzymatic expression, rats were divided into 5 study groups and administered either 3-methycholanthrene (3-MC, dissolved in corn oil, 50 mg/kg i.p. daily, for 2 days), phenobarbital (PB, dissolved in sterile isotonic saline, 80 mg/kg i.p. daily, for 3 days), dexamethasone (DX, dissolved in dimethyl sulfoxide/corn oil 1:1, 50 mg/kg i.p. daily, for 4 days), cyclosporine (CY, 30 mg/kg gavage in corn oil, daily, for 6 days) or paraquat (PQ, dissolved in sterile isotonic saline, 25 mg/kg i.p. daily, for 2 days). Rats were sacrificed by decapitation and the brains were rapidly removed and placed in an ice-cold isolation medium (0.32M sucrose, 1 mM EDTA, 10 mM Tris-HCl, pH 7.4). The lateral choroid plexus were located with a binocular microscope and sampled after ventrolateral incision of the brain. For enzymatic assessment, choroid plexus were homogenized in 10 volumes of isolation medium using a manual glass-glass Dounce-type homogenizer.

Enzymatic activities measurement

Measurement of 1-naphthol glucuronidation: conjugation assays were performed using a reverse phase chromatographic column (LiChrosorb RP-18: 5µm, Merck), with acetonitrile / water / acid acetic (25 / 74.5 / 0.5, v/v) as a mobile phase [10]. The NADPH-cytochrome P450 reductase activity in rat choroids plexus was measured at 25oC and =450 nm, with 10 μM cytochrome c as the substrate, in a 330 mM potassium phosphate 0,1 mM EDTA, pH 7,40. The reaction was started by the addition of NADPH at a final concentration of 46 μM [11]. Protein was measured following the method of Lowry et al. [12], using bovine serum albumin as a standard.



Reverse transcription-polymerase chain reaction (RT-PCR) 

Total tissular RNA was isolated and purified from the choroid plexus according to a microscale method using RNAXEL kit (Eurobio, Les Ulis, France). Complementary DNA was synthesized from RNA samples and for the study of the expression of both UGT1A6 and NADPH-cytochrome P450 reductase genes we used a semi-quantitative multistandard RT-PCR method, that consists in mixing the total RNA samples extracted from rat tissues with a constant amount of total RNA (serving as external standard) prepared from mouse cerebral tissue, which brought both -actin and UGT1A6 sequences [10,11].

Statistical analysis

Data were expressed as mean SD; Student t test was used to assess the significance of differences: p < 0.05 was considered as significant.
RESULTS AND DISCUSSION

The present study aimed to determine whether a broader scale of drug metabolizing enzyme inducers regulate glucuronidation and reductasic expression in rat choroids plexus. Five groups of chemicals that are known to regulate phase I and II drug metabolizing enzymes were administered to male Sprague-Dawley rats. By the use of specific primers toward mRNA encoding the rat liver UGT1A6 isoform, we were able to detect its constitutive and inducible expression in choroid plexus (Figure 1).






Figure 1

Representative electrophoresis and quantitative analysis of PCR products from UGT1A6 and -actin mRNA in the rat choroid plexus isolated after an “in vivo” treatment with either: 3-methylcholanthrene (3-MC), phenobarbital (PB), dexamethasone (DX), cyclosporine (CY) or paraquat (PQ). C, control; M, ladder of 1 Kb- DNA strands. Results are given as mean + SD, n=10; n- number of animals

* Significantly different from control rats (p < 0.001, Student t-test).

The expression of UGT1A6 mRNA was significantly increased 2,3 times and 2,1 times by 3-MC and PQ treatment respectively. In contrast, no regulation was observed with PB, DX or CY treatment.

Moreover, additional assays performed comparatively on mRNA isolated from brain parenchyma and choroid plexus confirmed the lack of induction of UGT1A6 at cerebral level (figure 2).




Figure 2

Representative electrophoresis and quantitative analysis of PCR products from UGT1A6 and -actin mRNA in the rat brain parenchyma and choroid plexus isolated after an “in vivo” treatment with paraquat (PQ). M, ladder of 1 Kb- DNA strands

Results are given as mean + SD, n=10; n- number of animals.


Besides, by the use of specific primers, we evidenced here for the first time the presence in rat choroids plexus the constitutive expression of the NADPH-cytochrome P450 reductase (figure 3). Enzyme mRNA expression remained unchanged in the isolated choroids plexus, in these experimental conditions (figure 3).



Figure 3

Representative electrophoresis and quantitative analysis of PCR products from NADPH-cytochrome P450 reducatse (410 bp) and -actin (220 bp) mRNA in rat choroid plexus isolated after an in vivo treatment with either: 3-methylcholanthrene (3-MC), phenobarbital (PB), dexamethasone (DX), cyclosporine (CY) or paraquat (PQ). C, control; M, ladder of 1 Kb- DNA strands. The relative levels of amplified cDNA were determined by densitometry; A P450 and A beta actin are absolute values of P450 reductase mRNA and -actin mRNA

Results are given as mean + SD, n=10; n- number of animals


1-naphthol can be considered in brain structures as a preferential substrate of UGT1A6. We measured 1-naphthol--D-glucuronide formation in choroid plexus. With regard to enzymatic activities, UGT1A6 was significantly increased (3 times) in 3-MC treated rats and in PQ treated rats (2 times) in correlation with mRNA modulation. While, no significant changes were recorded for the reductase activity (Table I).

Until now, the mainly expressed isoform of UDP-glucuronosyltransferases (UGTs) in the brain seems to be the UGT1A6, which conjugates a variety of planar phenolic xenobiotc compounds such as 1-naphthol, 4-nitrophenol, 4-methylumbelliferone, paracetamol, [13], and endogenous substrates such as serotonin [14].


Table I


1-Naphthol UGT1A6 and NADPH-cytochrome P450 reductase enzymatic activities in rat choroid plexus isolated after an in vivo treatment with either: 3-methylcholanthrene, phenobarbital, dexamethasone, cyclosporine or paraquat.


Treatment

1-Naphthol UGT1A6 specific activity

(nmoles/ hour x mg. protein)



NADPH-cytochrome P450 reductase specific activity

(nmoles cytochrome c reduced/ minute x mg. protein)



Control

115.32 ± 24.17

24.55 ± 8.27

3-Methylcholanthrene

354.25 ± 85.44 *

25.08 ± 7.17

Phenobarbital

127.15 ± 40.11

26.12 ± 9.85

Dexamethasone

110.45 ± 29.08

25.58 ± 10.5

Cyclosporine

125.87 ± 34.49

23.66 ± 7.85

Paraquat

257.51 ± 49.66 *

26.54 ± 9.92

Results are given as mean + SD, n=10; n- number of animals.

* Significantly different from control rats (p < 0.001, Student t-test).


The glucuronidation process, displays a regional and cellular heterogeneity, with high levels in the cerebral structures forming either blood-brain- and cerebrospinal fluid –brain interfaces [4,15]. At cellular level, the 1-naphthol UGT1A6 specific activity was 10-fold higher in astrocytes, as compared with neurons and endothelial cerebrovascular cells [16]. A high expression was also demonstrated in the olfactory bulb, which is a possible way of access for numerous xenobiotics, among them drugs and neurotoxicants. Moreover, studies regarding the age-related changes in rat brain and olfactory bulb UGT1A6 showed that 1-naphthol glucuronidation increased significantly during aging, without significant change in the messenger RNA (mRNA) levels encoding this isoform [6]. Besides the existence of UGT1A6 isoform, recent works demonstrated the presence of the UGT2A1 mRNA expression in the olfactory bulb [8]. This isoform, also called olfactory UGT isoform was initially thought to be expressed only in the nasal epithelium. All these data support the concept of a metabolic barrier located located at these sites.

NADPH-cytochrome P450 reductase is formed in all tissues that display cytochrome P450- mediated drug metabolism and/or heme oxygenase activities. Nevertheless, this reductase displays relatively high activities in isolated cerebral microvessels and catalyses the monoelectronic reduction of some drugs in cerebral preparation [9].



The use of powerful mRNA expression measurements by RT-PCR techniques definitely established the expression of UGT1A6 and NADPH-cytochrome P450 reductase in both the brain and choroids plexus. Moreover, the use of semi-quantitative RT-PCR allowed us to show that UGT1A6 mRNA expression increased in the choroid plexus after an “in vivo” treatment of animals by 3-MC and PQ but no changes were observed after PB, DX and CY administration, in good accordance with some of the previous observations concerning the effect of a treatment with these molecules on 1-naphthol glucuronidation in the brain and in choroids plexus [3].

Recent findings show that rat choroid plexus, as well as human choroids plexus from both fetal and adult brains, displayed high glutathione-S-transferase activities [17].


CONCLUSIONS

The present results suggest that xenobiotics could modulate the biotransformation of exogenous and/or endogenous compounds in the choroids plexus, and underline the role of UGTs in the maintenance of brain homeostasis.



Also, choroids plexus affords a metabolic protection to the brain against xenobiotics. Both phase I and II drug metabolism activities could represent either an obstacle or a tool for the administration of pharmacological agents to the brain and should prevent potentially harmful drugs from readily gaining access to CSF and brain.
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