Role of Two Efflux Proteins, ABCB1 and ABCG2, in Blood-Brain Barrier Transport of Bromocriptine in a Murine Model of MPTP-Induced Dopaminergic Degeneration

PURPOSE
MPTP-induced dopaminergic degeneration is an experimental model commonly used to explore Parkinson's disease. Cerebral drug transport by ABC transporters in MPTP models has never been reported.


METHODS
We have investigated the transport of bromocriptine through the blood-brain barrier (BBB) in a MPTP model to understand the influence of the dopaminergic degeneration on ABCB1 and ABCG2.


RESULTS
We have shown that in MPTP treated mice, bromocriptine is widely distributed to brain (2.3-fold versus control, p less than 0.001) suggesting either disruption of BBB or alteration of active efflux of the drug. In situ brain perfusion of [14C]- sucrose and [3H]-inulin did not evidenced a BBB disruption. Studies of ABCB1 and ABCG2 activity showed that MPTP intoxication did not alter their functionality. Conversely, ABCG2 expression studied on brain capillaries from MPTP-treated mice was decreased (1.3-fold, p less than 0.05) and ABCB1 expression increased (1.43-fold, p less than 0.05) as an off-setting of brain transport.


CONCLUSIONS
These data demonstrate that MPTP intoxication does not alter the BBB permeability. However, bromocriptine brain distribution is increased in MPTP animals. Hence, MPTP may interact with another transport mechanism such as uptake and/or other efflux transporters. Inflammation and Parkinson's-like lesions induced by MPTP intoxication could lead to modification of drug pharmacokinetics and have clinical consequences, such as neurotoxicity.


INTRODUCTION
Parkinson's disease (PD) is primarily a neurodegenerative disorder characterized by a large loss of nigral dopaminergic neurons with dopamine depletion and formation of Lewy bodies.PD treatment consists in the administration of dopamine receptor agonists and levodopa, as dopamine does not cross the bloodbrain barrier (BBB).It has been postulated that exogenous toxicants, including pesticides, might be involved in the etiology of PD (1).Several in vivo models of dopaminergic degeneration, using neurotoxins such as rotenone, 6hydroxydopamine (6-OHDA) or MPTP, have been developed to study PD (2-4).MPTP, and its metabolite MPP + , are potent and specific nigral toxins able to inhibit the complex I of the mitochondrial electron transport chain (5).The model of MPTP intoxication is an interesting model for the earliest phase of PD where clinical symptoms appear and neuroprotective therapy should be introduced.
Drugs used for PD treatment need to cross the brain capillary wall to be active.Only small molecules with high lipid solubility and low molecular mass (<400 Da) cross the BBB by passive process in pharmacologically significant amounts.Many other drugs are transported across the BBB by an active transport.Conversely, cerebral efflux pumps are expressed on the luminal side of BBB endothelial cells and are responsible for the extrusion of their substrates from endothelial cells back to the blood circulation.
Levodopa crosses BBB through LAT1 (Large neutral Amino-acid Transporter) and is converted in dopamine within the brain by aromatic amino acid decarboxylase (AAAD).While Soares-da-Silva et al. have shown that levodopa is excluded from brain capillary endothelial cells by ABCB1 (7, 8), other authors showed that levodopa does not interfere with ritonavir uptake by ABCB1 expressing cells (9).Bromocriptine, an antiparkinsonian drug which is also a substrate of ABCB1, is weakly transported in cerebral tissues and low concentrations are detected in mice brain (10).Shiraki et al., as well as Orlowski et al. have also shown that bromocriptine interacts with ABCB1 in vitro (11,12).Uhr et al. have shown that budipine, another dopamine agonist, was transported out of the brain by ABCB1 (13).ABCG2 also plays a role in drug efflux of many drugs but interactions between ABCG2 and bromocriptine have not been described.
Multidrug resistance occurs in several diseases such as inflammation, cancer or epilepsy (14-16).Pharmacoresistance is often related to ABCB1 overexpression and has been widely described in these diseases (17,18).However, few data are available on the expression and function of ABCB1 in PD.Alterations of BBB associated with a compensatory increase of ABCB1 expression were observed in mice exposed to the neurotoxin 6-hydroxydopamine, suggesting that brain distribution of drugs would be modified (19).Hence, the aim of this study was to investigate the transport of bromocriptine through the BBB and understand the influence of dopaminergic degeneration on its efflux by ABCB1 and ABCG2 functionality, in a model of MPTP-induced Parkinsonism syndrome.MPTP can impair BBB integrity.As large amounts of this neurotoxin are required to achieve neurodegeneration, we also had to explore BBB integrity.

Acute MPTP intoxication protocol
The MPTP-treated groups received intraperitoneal (ip) injections of MPTP every 2 hours (4 x 15 mg.kg -1 ) and control-groups received saline solution every 2 hours (4 times, ip) with equivalent volumes at day 0. All injections were given in a volume of 250 µL/25g of body weight.This experimental model was previously validated by Rousselet et al. (20) who showed that MPTP injections led to a loss of nigral dopaminergic neurons, a reduction in striatal dopamine content and a behavioural disturbance.As reported by the authors, in this murine model, all animals exhibit an homogeneous and severe loss (90%) of dopamine content (20).All experiences were conducted on day 7, since MPTP and metabolites were completely excreted and neurotoxicity by close contact was void (21).Blood was collected by intracardiac puncture after anesthesia with isoflurane.The brain was collected after euthanasia.Plasma and brain samples were stored frozen at -80°C until analysis.

Transport study of bromocriptine through the BBB
BCT was administered to MPTP-treated mice and saline-treated mice, at the dose of 20 mg.kg -1 (water:methanol, 95:5, v/v).Studies were repeated on animals previously treated with a ABCB1 inhibitor, PSC833 at the dose 15 mg.kg -1 (PEG300:ethanol, 80:20, v/v) (Table 1) in order to inhibit specifically ABCB1 activity (22).In each group (n=9), BCT was administered by ip route 30 min after PSC833 injection, corresponding with its plasmatic T max .The same schedule was followed for the control group.
Brain and plasma were collected 1 hour after BCT injection.BCT concentration was determined in brain and plasma by HPLC using an analytical method validated in our laboratory and previously reported (10).Briefly, the analytical separation was achieved using a Lichrospher ® 100 RP18 column (150 x 4.6 mm, 5 µm) with a fluorimetric detection ( ex : 330 nm;  em : 405 nm) and a mobile phase composed of water with 0.2% diethylamine (pH adjusted to pH3.0) and acetonitrile (70:30, v/v) running at a flow rate of 1 mL.min -1 .BCT was extracted from brain and plasma using MCX ® OASIS cartridges (60 mg, 3 mL) (Waters, Millipore, Saint Quentin, France) with 1 mL of methanol/ammonia (97.5:2.5;v/v).
Brain tissue total BCT was corrected for vascular contamination using the following formula: where Q total brain is the total BCT amount in brain, including cerebral capillaries and cerebral tissue, Q cap is the BCT amount in cerebral capillaries and Q tissue is the BCT amount in brain tissue.Q cap was calculated as: where V vasc is the brain vascular volume [13 µL.g -1 of cerebral tissue ( 23)] and C plasma is the BCT plasma concentration.Q cap , the BCT amount in cerebral capillaries.C tissue was calculated as: C tissue = Q tissue /total brain weight where C tissue is the BCT concentration in brain tissue and Q tissue is the BCT amount in brain tissue.C tissue was used to calculate the brain/plasma ratios Kp according to Kp = C tissue / Cplasma These ratios representing cerebral transport were then calculated and compared.

Determination of the blood-brain barrier integrity by in situ brain perfusion
MPTP-treated mice (n=4) and control mice (n=3) were anesthetized by ip injection of a mixture of xylazine (Bayer, Puteaux, France) and ketamine (Panpharma, Fougères, France) at 8/140 mg kg −1 .Briefly, the common carotid artery was ligated on the heart side.The external carotid was ligated rostral to the occipital artery at the level of the bifurcation of the common carotid artery.Then, the right common carotid was catheterized with polyethylene tubing (0.30 mm i.d.× 0.70 mm o.d.; Folioplast, Sarcelles, France) filled with heparin (25 IU.mL −1 ).The thorax of the mouse was opened and the heart was cut.Perfusion was immediately started (flow rate: 2.5 mL.min −1 ) with the syringe containing the perfusion fluid placed in an infusion pump (Harvard pump PHD 2000, Harvard Apparatus, Holliston, MA, USA) and connected to the catheter.The perfusion fluid consisted of bicarbonate-buffered physiological saline (mM): 128 NaCl, 24 NaHCO 3 , 4.2 KCl, 2.4 NaH 2 PO 4 , 1.5 CaCl 2 , 0.9 MgCl 2 and 9 D-glucose.The solution was gassed with 95% O2 and 5% CO2 for pH control (7.4) and warmed to 37°C in a water bath.Appropriate concentrations of compounds were added to the perfusate.Each mouse was perfused with [ 3 H]-inulin (0.35 µCi.mL −1 ) and [ 14 C]-sucrose (0.30 µCi.mL −1 ).Perfusion was terminated by decapitation at 60 s.The brain was removed from the skull and dissected out on ice.Each right cerebral hemisphere was placed in a tared vial and weighed.Aliquots of the perfusion fluid were also collected and weighed to determine tracer concentrations in the perfusate.Samples were digested in 1 mL of Soluène ® at 50°C and mixed with 10 ml of Ultima gold ® counting fluid (Perkin-Elmer Life and Analytical Sciences, Boston, USA).Radioactivity was determined by liquid scintillation counting on a Beckman LS 6000 TA counter (Beckman, Galway, Ireland).

Calculation of brain vascular volume (Vvasc)
The calculation of the brain vascular volume has previously been described (23,24).The brain vascular volume (Vvasc, µl.g -1 ) was assessed with [ 14 C]-sucrose and [ 3 H]-inulin, which do not cross the BBB during short exposure.
V vasc = X brain /C perf where X brain (dpm.g−1 ) is the amount of [ 3 H]-or [ 14 C]-compound in the right cerebral hemisphere, and C perf (dpm.µL−1 ) is the concentration of [ 14 C]sucrose or [ 3 H]-inulin in the perfusion fluid.

Investigation of ABCB1 and ABCG2 functionality
A well known and validated ABCB1 substrate, [ 3 H]-digoxin, was administered to MPTP-treated mice and saline-treated mice, at 0.5 mg/kg (1µCi/mice) to investigate ABCB1 activity.Plasma and brains were collected 6 hours after administration of [ 3 H]-digoxin.Dose and timing of sampling were extrapolated from previous published data : 6 hours were chosen as the equilibrium time of brain/plasma concentration ratio (22,25,26).
On the other hand, a ABCG2 substrate, [ 3 H]-prazosin, was administered to MPTP-treated mice and saline-treated mice, at 1 mg/kg (1µCi/mice) to investigate ABCG2 activity.Plasma and brains were collected 1 hour after administration of [ 3 H]-prazosin.
Samples were transferred to Ultima Gold ® counting fluid for plasma and Hionic Fluor ® for brains (Perkin-Elmer Life and Analytical Sciences, Boston, USA).Radioactivity was determined by liquid scintillation counting on a Beckman LS 6000 TA counter (Beckman, Galway, Ireland).Brain/plasma concentrations ratios were calculated: they represent the cerebral transfer of digoxin or prazosin.

Isolation of brain capillaries
Brain microvessels were isolated from mice brains by using the capillary depletion method of Triguero et al. (27) with slight modifications.After euthanasia, brains were removed from mice skulls and promptly immerged in ice-cold phosphate-buffered saline (PBS; Invitrogen Corporation, Cergy-Pontoise, France).They were then cleared of cerebellum, hind-and midbrain, superficial blood vessels, and meninx.Afterwards, half of the remaining cortex was gently homogenized in ice-cold Dulbecco's modified Eagles medium/fetal calf serum (DMEM/FCS; 90:10; v:v) (Invitrogen Corporation) by using a Teflon potter.The homogenates were next centrifuged at 1,000 g for 10 min.After elimination of the supernatant, pellets were homogenized in 5 mL of a 25% BSA solution and centrifuged again at 1,500 g for 20 min.Supernatants were thereafter removed and pellets were suspended in 1 mL of the same icecold mixture of DMEM/FCS and filtrated at 60 μm.Filtrates containing brain capillaries were centrifuged at 12,000 g for 45 min.Pellets of capillaries were washed and resuspended in icecold PBS.Microvessels suspensions were finally centrifuged at 12,000 g for 20 min.Pellets were immediately prepared for protein extraction and semi-quantification.
After 5 times of 10 min washes in TTBS, they were further incubated 1 hour at room temperature with anti-mouse horseradish peroxidase-conjugated antibody diluted to 1:10000 (Dako, Glostrup, Denmark).The membranes were washed 5 times for 10 min in TTBS and then probed using the Western Lightning chemiluminescence reagent (Perkin Elmer; Courtaboeuf, France).The intensity of bands was quantified using Scion Image (NIH, Scion Corporation, Bethesda, USA) and protein expression was normalized with β-actin.

Statistical analysis
All data are expressed as mean ± SEM.A non parametric Mann-Whitney test was used for statistical analysis.P<0.05 was considered statistically significant.

Determination of the BBB integrity by in situ brain perfusion
[ 14 C]-sucrose and [ 3 H]-inulin were used as markers of vascular space.The vascular volume was determined in the mouse brain after a 60second perfusion with buffer : V vasc of sucrose = 12.63±1.45µL.g -1 in CT mice (n=4) vs 12.85±0.44µL.g -1 in MPTP mice (n=4) and V vasc of inulin = 8.69±0.31µL.g - in CT mice vs 8.12±0.45µL.g -1 in MPTP mice.The vascular volumes obtained in the present study agree with previously reported values (23,24).[ 14 C]-sucrose V vasc were 1.4-fold higher than the [ 3 H]-inulin V vasc , which is consistent with the study of Cattelotte et al. in which [ 14 C]-sucrose V vasc was 1.2-fold higher than [ 3 H]-inulin V vasc (24).This discrepancy should be attributed to the lower molecular weight and higher permeability of sucrose relative to inulin (28).

ABCB1 and ABCG2 functionality
Digoxin was chosen as a substrate of ABCB1 in order to investigate the functionality of this efflux pump.We did not observe any significant difference in brain/plasma ratios between MPTPtreated mice and control mice (Table 2).
Prazosin was chosen as ABCG2 substrate to investigate its functionality.We did not observe any difference in brain/plasma ratios between MPTP-treated mice and control mice (Table 2).Inhibition of ABCB1 and ABCG2 activity was validated with PSC833 and GF120918, a specific inhibitor of ABCB1 and an inhibitor of ABCG2.We have shown that digoxin and prazosin brain transport were increased in PSC833 and in GF120918-treated animals (data not shown).
Hence, intoxication with MPTP did influence neither ABCB1 nor ABCG2 functionality.

ABCB1 and ABCG2 expression at the BBB
There was a difference in the expression of efflux transporters in cerebral capillaries of MPTPtreated mice compared to that of saline-treated mice: in brain capillaries from MPTP mice, ABCB1 expression was increased by 1.43-fold (p<0.05) when compared to that of control mice (Figure 2).On the other hand, ABCG2 expression was decreased by 1.3-fold when compared to that of control mice (Figure 3)

DISCUSSION
Our study shows that intoxication of mice with MPTP enhances brain distribution of BCT.In order to explain this phenomenon, we have hypothesized that (i) BBB permeability is increased by MPTP and/or (ii) that MPTP is responsible for an alteration of BCT efflux by transporters from the brain, resulting in its accumulation.
MPTP was used as a validated model of PD.Rousselet et al. have previously shown that MPTP-treated mice exhibit loss of dopaminergic neurons and dopamine content (20).In their study, they did not evidence any failure in intoxication.As we needed the whole brain to perfom drug (bromocriptine, prazosin and digoxine) or protein quantification, we could not check by immunohistochemistry to what extent were affected dopamine neurons.
As BBB permeability can influence brain diffusion of xenobiotics and may be disrupted by some toxics, we have investigated BBB integrity.BBB is characterized by tight junctions and adhesin molecules, between brain microvessel endothelial cells, that prohibit the crossing of xenobiotics (29).Brain vascular permeability is increased by cerebral ischemia, infections or inflammatory diseases like cerebral malaria (30).Experimental models of cerebral malaria after infection of C57BL/6N mice with Plasmodium berghei K173 have evidenced an increase of vascular permeability and cerebral oedema, permitting to epileptogene factors, to cross the BBB (30-32).MPTP was also responsible for brain inflammation that led to a second-time microglial activation which generated neuronal destruction (33).Studies using deficient mice in COX-2 gene, inductible NO synthase gene or TNF receptors, all of them playing a role in inflammatory process, showed that blockade of microglial activation has a neuroprotective effect against MPTP toxicity (34-36).We estimated the brain vascular volume of MPTP mice by measuring the brain volume of distribution of two vascular space markers, [ 3 H]-inulin and [ 14 C]sucrose, using the in situ brain perfusion technique.Since our BCT experiments were performed 7 days after the MPTP intoxication, we have investigated the BBB integrity at day 7 after the MPTP administration.This experiment has confirmed that the BBB was not damaged 7 days after mice were intoxicated with MPTP.But we cannot exclude that a transitory BBB disruption occurs between the day 0 and 7. Cerebral alterations in a MPTP model have been studied by Muramatsu et al. during 7 days after intoxication (37).They have shown that the neuronal NO synthetase was markedly decreased in the substantia nigra 3 and 7 days after MPTP treatment.In another rat model of glial cell degeneration, Guerin et al. have demonstrated that BBB integrity was largely re-established within 6 days after intoxication by dinitrobenzene (38).In conclusion, the increase of BCT cerebral uptake observed in our experiments could not be the outcome of an alteration of BBB integrity.The second hypothesis to explain the increase in BCT cerebral diffusion in MPTP mice is an alteration of functionality and/or expression of efflux transporters involved in BCT cerebral transport.We have already reported elsewhere that BCT is transported by ABCB1 (10).The accumulation of BCT in brain of PSC833 pretreated mice confirmed this result.However, ABCB1 inhibition by PSC833 does not modify brain distribution of BCT in MPTP-treated mice, suggesting that in these animals ABCB1 is not involved in BCT transport.Furthermore, digoxin transport is not modified by MPTP intoxication, showing that in MPTP treated animals, ABCB1 function is not altered.
In our experiments, ABCB1 expression was increased in MPTP animals.This increase could be explained by the inflammatory process (39).Previous works reported that MDR1 transcription could be activated by a transcriptional regulator nuclear factor for Interleukin-6 (NF-IL6), a member of the C/EBPβ family of transcription factors.NF-IL6 is inductible by stress or inflammation and would be able to induce MDR1 expression (40).ABCB1 expression is also up-regulated by the steroid xenobiotic receptor SXR (pregnant X receptor, PXR in rodents) which is a member of a family of ligand-activated nuclear receptor that regulate expression of drug metabolizing enzymes and transporters.This activation was demonstrated in rat brain capillaries where PXR ligands enhanced ABCB1 expression (41).Relations between MPTP or MPP + and SXR/PXR have not been described in literature.However, MPTP or MPP + could be a PXR-ligand or another nuclear receptor ligand which up-regulates ABCB1 expression in brain capillaries.
The increase of ABCB1 expression is not followed by an increase of ABCB1 functionality as digoxin transport is not altered by MPTP intoxication.Examples of lack of correlation between ABCB1 expression and ABCB1 activity have already been reported in many assays.For instance, Regina et al. (42) have demonstrated an increase of ABCB1 functionality without any increase of ABCB1 expression after treatment of cerebral capillaries endothelial cells GPNT with dexamethasone.At the BBB level, the decrease of ABCB1 functionality without modification of the protein expression was also observed by Mandi et al. (43) on human BB19 cerebral capillary endothelial cells, after TNF-α treatment.MPTP could up-regulate ABCB1 expression but leads to the synthesis of a non functional protein.Conversely, Zhao et al. (44) have found an increase of ABCB1 expression with a decrease of its functionality in mice.The discrepancy between ABCB1 expression and functionality could be explained by a decrease in the ATP content in the brain or by the accumulation of endogenous ABCB1 substrates in plasma.In their paper, Zhao et al. retain the second hypothesis.In the same way, Staal et al. (45) have reported that MPP + is extruded from mice brain by ABCB1 but results have not been confirmed in vitro.However, a competition between MPP + , the active metabolite of MPTP and BCT could not explain BCT brain accumulation as on day 7, all MPTP and MPP + should be eliminated.ABCB1 protein could also been overexpressed in a cytoplasmic pool but not addressed at the luminal side of endothelial cells and, so not functional.
Hence, the increase of cerebral uptake of BCT does not result from an alteration of transport via ABCB1.Recent studies have demonstrated that ABCG2 exerts an impact on drug absorption and disposition (46).We have demonstrated that ABCG2 function is not altered by the dopaminergic neurodegeneretion but its cerebral expression decreases following MPTPinduced intoxication.Literature doesn't report any case of discrepancy between ABCG2 expression and functionality.
Our studies showed that ABCB1 expression was increased whereas ABCG2 expression was decreased.Compensation between efflux transporters expression has already been described in vivo, in deficient mouse models : in mutant CF-1 mdr1a(-/-) mice, the lack of in cerebral ABCB1 is associated with an increase of ABCG2 mRNA at the BBB compared to wildtype mice.
However, many other proteins belonging to the ABC transporters family are present at the BBB such as MRPs (47) or uptake transporters (organic anion transporter polypeptid Oatp2, Oatp14, LAT1) (48).We suppose that high levels of the neurotoxin MPTP, necessary to achieve the neurodegenerative model, could also alter other transporters implicated in cerebral uptake or efflux.
In conclusion, our assays show that, in a model of dopaminergic neurodegeneration, BCT brain diffusion is increased.This phenomenon which could lead to central toxicity of BCT and increase of adverse events in patients is not related to ABCB1 or ABCG2 dysfunction.Furthermore, our work gives one more example of discrepancy between ABCB1 expression and functionality.

Table 2 .
Brain/plasma ratios of probe substrates (mean ± SEM) in ABCB1 and ABCG2 functionality study, after single ip injection of substrates in control mice and MPTP-treated mice.Effect of MPTP-induced toxicity on ABCG2 expression.The values represent mean intensities normalized with β-actin in control group (CT, n=7) and MPTP-treated group (MPTP, n=7), * p<0.05.mean ± SEM.