A simple novel approach for detecting blood-brain barrier permeability using GPCR internalization.

AIMS: Impairment of blood-brain barrier (BBB) is involved in numerous neurological diseases from developmental to aging stages. Reliable imaging of increased BBB permeability is therefore crucial for basic research and preclinical studies. Today, the analysis of extravasation of exogenous dyes is the principal method to study BBB leakage. However, these procedures are challenging to apply in pups and embryos and may appear difficult to interpret. Here we introduce a novel approach based on agonist-induced internalization of a neuronal G protein-coupled receptor widely distributed in the mammalian brain, the somatostatin receptor type 2 (SST2). METHODS: The clinically approved SST2 agonist octreotide (1 kDa), when injected intraperitoneally does not cross an intact BBB. At sites of BBB permeability, however, OCT extravasates and induces SST2 internalization from the neuronal membrane into perinuclear compartments. This allows an unambiguous localization of increased BBB permeability by classical immunohistochemical procedures using specific antibodies against the receptor. RESULTS: We first validated our approach in sensory circumventricular organs which display permissive vascular permeability. Through SST2 internalization, we next monitored BBB opening induced by magnetic resonance imaging-guided focused ultrasound in murine cerebral cortex. Finally, we proved that after intraperitoneal agonist injection in pregnant mice, SST2 receptor internalization permits analysis of BBB integrity in embryos during brain development. CONCLUSIONS: This approach provides an alternative and simple manner to assess BBB dysfunction and development in different physiological and pathological conditions.

New diketopiperazines as vectors for peptide protection and brain delivery: Synthesis and biological evaluation.

New strategies allowing the transfer of molecules, especially peptides, through the blood-brain barriers are a major pharmacological challenge for the treatment of brain diseases. The present study aims at evaluating in vivo the cerebral bioavailability of carrier systems, based on small and functionalizable 2,5-diketopiperazine (DKP) motifs. We studied 2 different cyclo(Lys-Lys) DKP scaffolds alone and a cyclo(Lys-Gly) DKP carrier bearing as peptide model, the tau protein hexapeptide VQIVYK sequence. The different carrier systems were synthesized and radiolabeled using one of the free domains. The stability, biodistribution, and ability to cross blood-brain barrier were investigated in vivo in mice for 99m Tc-DKP scaffolds, 99m Tc-HVQIVYK peptide alone, and 99m Tc-DKP-VQIVYK. 125 I-labelled bovine serum albumin was used as negative control for brain uptake. Both radiolabeled DKPs scaffolds and 99m Tc-DKP-VQIVYK showed a high stability, while peptide 99m Tc-HVQIVYK alone was quickly degraded in vivo. The presence of 99m Tc-DKPs scaffolds and 99m Tc-DKP-VQIVYK was observed in the ventricular and subarachnoid spaces and to a lower extent in the brain parenchyma up to 45 minutes post-injection in mice. This work highlights the potentiality of DKP scaffolds as vectors to transport peptides into the brain by limiting proteolysis and favoring cerebral bioavailability.

Physiology of blood-brain interfaces in relation to brain disposition of small compounds and macromolecules.

The brain develops and functions within a strictly controlled environment resulting from the coordinated action of different cellular interfaces located between the blood and the extracellular fluids of the brain, which include the interstitial fluid and the cerebrospinal fluid (CSF). As a correlate, the delivery of pharmacologically active molecules and especially macromolecules to the brain is challenged by the barrier properties of these interfaces. Blood-brain interfaces comprise both the blood-brain barrier located at the endothelium of the brain microvessels and the blood-CSF barrier located at the epithelium of the choroid plexuses. Although both barriers develop extensive surface areas of exchange between the blood and the neuropil or the CSF, the molecular fluxes across these interfaces are tightly regulated. Cerebral microvessels acquire a barrier phenotype early during cerebral vasculogenesis under the influence of the Wnt/ß-catenin pathway, and of recruited pericytes. Later in development, astrocytes also play a role in blood-brain barrier maintenance. The tight choroid plexus epithelium develops very early during embryogenesis. It is specified by various signaling molecules from the embryonic dorsal midline, such as bone morphogenic proteins, and grows under the influence of Sonic hedgehog protein. Tight junctions at each barrier comprise a distinctive set of claudins from the pore-forming and tightening categories that determine their respective paracellular barrier characteristics. Vesicular traffic is limited in the cerebral endothelium and abundant in the choroidal epithelium, yet without evidence of active fluid phase transcytosis. Inorganic ion transport is highly regulated across the barriers. Small organic compounds such as nutrients, micronutrients and hormones are transported into the brain by specific solute carriers. Other bioactive metabolites, lipophilic toxic xenobiotics or pharmacological agents are restrained from accumulating in the brain by several ATP-binding cassette efflux transporters, multispecific solute carriers, and detoxifying enzymes. These various molecular effectors differently distribute between the two barriers. Receptor-mediated endocytotic and transcytotic mechanisms are active in the barriers. They enable brain penetration of selected polypeptides and proteins, or inversely macromolecule efflux as it is the case for immnoglobulins G. An additional mechanism specific to the BCSFB mediates the transport of selected plasma proteins from blood into CSF in the developing brain. All these mechanisms could be explored and manipulated to improve macromolecule delivery to the brain.

[Biological roles of trace elements in the brain with special focus on Zn and Fe]. / Rôles biologiques des éléments traces dans le cerveau--exemples du Zn et du Fe.

INTRODUCTION: Many metals like iron (Fe), copper (Cu) or zinc (Zn) fulfil various essential biological functions and are thus vital for all living organisms. For instance, they play important roles in nervous tissue, participating in a wide range of processes such as neurotransmitter synthesis, myelination or synaptic transmission. STATE OF THE ART: As in other tissues, brain cells tightly control the concentration of metals but any excess or deficit can lead to deleterious responses and alter cognitive functions. Of note, certain metals such as Zn, Fe or Cu accumulate in specific brain structures over lifespan and several neurodegenerative diseases are associated with a dysregulation of the homeostatic mechanisms controlling the concentration of these cations. CONCLUSION AND PERSPECTIVES: This review will address some of the cellular and molecular processes controlling the entry and distribution of selected metals (mainly Zn and Fe) in the brain, as well as their roles in synaptic transmission, in the pathogenesis of some neurologic diseases such as Parkinson's disease and Alzheimer's disease, and their impact on cognitive functions.

Blood-brain interfaces and cerebral drug bioavailability.

The low cerebral bioavailability of various drugs is a limiting factor in the treatment of neurological diseases. The restricted penetration of active compounds into the brain is the result of the same mechanisms that are central to the maintenance of brain extracellular fluid homeostasis, in particular from the strict control imposed on exchanges across the blood-brain interfaces. Direct drug entry into the brain parenchyma occurs across the cerebral microvessel endothelium that forms the blood-brain barrier. In addition, local drug concentration measurements and cerebral imaging have clearly shown that the choroid plexuses - the main site of the blood-cerebrospinal fluid (CSF) barrier - together with the CSF circulatory system also play a significant role in setting the cerebral bioavailability of drugs and contrast agents. The entry of water-soluble therapeutic compounds into the brain is impeded by the presence of tight junctions that seal the cerebral endothelium and the choroidal epithelium. The cerebral penetration of many of the more lipid-soluble molecules is also restricted by various classes of efflux transporters that are differently distributed among both blood-brain interfaces, and comprise either multidrug resistance proteins of the ATP-binding cassette superfamily or transporters belonging to several solute carrier families. Expression of these transporters is regulated in various pathophysiological situations, such as epilepsy and inflammation, with pharmacological consequences that have yet to be clearly elucidated. As for brain tumour treatments, their efficacy may be affected not only by the intrinsic resistance of tumour cells, but also by endothelial efflux transporters which exert an even greater impact than the integrity of the endothelial tight junctions. Relevant to paediatric neurological treatments, both blood-brain interfaces are known to develop a tight phenotype very early on in postnatal development, but the developmental profile of efflux transporters still needs to be assessed in greater detail. Finally, the exact role of the ependyma and pia-glia limitans in controlling drug exchanges between brain parenchyma and CSF deserves further attention to allow more precise predictions of cerebral drug disposition and therapeutic efficacy.

[The choroid plexuses: a dynamic interface between the blood and the cerebrospinal fluid]. / Les plexus choroïdes: une interface dynamique entre sang et liquide cephalo-rachidien.

The choroid plexuses form one of the interfaces that control the brain microenvironment by regulating the exchanges between the blood and the central nervous system. They appear early during brain development. Originating from four different areas of the neural tube, they protrude into the ventricular system of the brain. The choroidal mechanisms involved in the control of brain homeostasis include the structural properties of the epithelial cells that restrict diffusional processes, as well as specific exchange and secretion mechanisms. In addition to the anatomical and histological organization of the choroidal tissue, this review describes the mechanism of cerebrospinal fluid secretion which is the most studied function of the choroid plexus. Experimental evidence for an implication of the choroid plexuses in neuroprotective mechanisms and in the supply of biologically active polypeptides to the brain are also reviewed.

Melatonin reduces excitotoxic blood-brain barrier breakdown in neonatal rats.

The blood-brain barrier (BBB) is a complex structure that protects the central nervous system from peripheral insults. Understanding the molecular basis of BBB function and dysfunction holds significant potential for future strategies to prevent and treat neurological damage. The aim of our study was (1) to investigate BBB alterations following excitotoxicity and (2) to test the protective properties of melatonin. Ibotenate, a glutamate analog, was injected intracerebrally in postnatal day 5 (P5) rat pups to mimic excitotoxic injury. Animals were than randomly divided into two groups, one receiving intraperitoneal (i.p.) melatonin injections (5mg/kg), and the other phosphate buffer saline (PBS) injections. Pups were sacrificed 2, 4 and 18 h after ibotenate injection. We determined lesion size at 5 days by histology, the location and organization of tight junction (TJ) proteins by immunohistochemical studies, and BBB leakage by dextran extravasation. Expression levels of BBB genes (TJs, efflux transporters and detoxification enzymes) were determined in the cortex and choroid plexus by quantitative PCR. Dextran extravasation was seen 2h after the insult, suggesting a rapid BBB breakdown that was resolved by 4h. Extravasation was significantly reduced in melatonin-treated pups. Gene expression and immunohistochemical assays showed dynamic BBB modifications during the first 4h, partially prevented by melatonin. Lesion-size measurements confirmed white matter neuroprotection by melatonin. Our study is the first to evaluate BBB structure and function at a very early time point following excitotoxicity in neonates. Melatonin neuroprotects by preventing TJ modifications and BBB disruption at this early phase, before its previously demonstrated anti-inflammatory, antioxidant and axonal regrowth-promoting effects.

Choroid plexus in the central nervous system: biology and physiopathology.

Choroid plexuses (CPs) are localized in the ventricular system of the brain and form one of the interfaces between the blood and the central nervous system (CNS). They are composed of a tight epithelium responsible for cerebrospinal fluid secretion, which encloses a loose connective core containing permeable capillaries and cells of the lymphoid lineage. In accordance with its peculiar localization between 2 circulating fluid compartments, the CP epithelium is involved in numerous exchange processes that either supply the brain with nutrients and hormones, or clear deleterious compounds and metabolites from the brain. Choroid plexuses also participate in neurohumoral brain modulation and neuroimmune interactions, thereby contributing greatly in maintaining brain homeostasis. Besides these physiological functions, the implication of choroid plexuses in pathological processes is increasingly documented. In this review, we focus on some of the novel aspects of CP functions in relation to brain development, transfer of neuro-humoral information, brain/immune system interactions, brain aging, and cerebral pharmaco-toxicology.

In vitro evidence that beta-amyloid peptide 1-40 diffuses across the blood-brain barrier and affects its permeability.

Beta amyloid peptides are major insoluble constituents of amyloid fibrils in senile plaques and cerebrovascular deposits, both characteristic of Alzheimer disease (AD). Low concentrations of soluble forms of amyloid peptides are also present in normal CSF. We previously demonstrated that the 40 amino acid form of soluble beta-amyloid peptide (sAbeta) is rapidly cleared from rat CSF into blood. Herein we hypothesized that a saturable, outwardly directed flux of this peptide occurs at the blood-brain barrier (BBB) and tested whether supraphysiological (possibly pathological) concentrations of sAbeta could alter the permeability of this barrier to a paracellular tracer, polyethylene glycol (PEG). Using an in vitro model of BBB, we showed that influx and efflux of sAbeta were equal, modest (60%-160% greater than that of PEG), and not saturable. These observations suggest that sAbeta moved across the monolayer by a diffusional process, and not via a transporter. PEG flux was doubled immediately after the luminal concentration of cold sAbeta was raised to 5 microM, and was doubled 150 min after the abluminal concentration of sAbeta was increased to 5 microM. Pathological elevations of sAbeta concentration in plasma or brain interstitial fluid may, therefore, alter the permeability of brain capillaries in vivo.

A simple method for evaluation of superoxide radical production in neural cells under various culture conditions: application to hypoxia.

To evaluate the potential deleterious influence of oxygen-derived free radicals following hypoxia in a model of primary culture of neurons obtained from the fetal rat brain, superoxide radicals were measured as a function of time in the extracellular medium. Neuronal cells were grown for 8 days in the presence or absence of serum, then incubated in a buffered Krebs-Ringer solution containing 60 microM acetyl-cytochrome c. The rate of superoxide radical formation was quantified spectrophotometrically by measuring the specific reduction of acetyl-cytochrome c. Under normoxic conditions (95% air-5% CO2), basal production of superoxide that increased with time was recorded. It was significantly more pronounced in cells grown in serum-free medium. Under both culture conditions, acute hypoxia (95% N2-5% CO2) for 6 h increased superoxide radical amounts in the extracellular medium, and they were still enhanced 3 h after reoxygenation. The addition of superoxide dismutase to the incubating medium abolished the detection of superoxide radicals. The present study describes a new reliable method for superoxide radical measurement in cells in vitro and demonstrates hypoxia/reoxygenation-induced overproduction of superoxide in cultured neurons that may account for cell injury.

Nicotine raises the influx of permeable solutes across the rat blood-brain barrier with little or no capillary recruitment.

Nicotine (1.75 mg/kg s.c.) was administered to rats to raise local CBF (lCBF) in various parts of the brain, test the capillary recruitment hypothesis, and determine the effects of this increase in lCBF on local solute uptake by brain. lCBF as well as the local influx rate constants (K1) and permeability-surface area (PS) products of [14C]antipyrine and [14C]-3-O-methyl-D-glucose (3OMG) were estimated by quantitative autoradiography in 44 brain areas. For this testing, the finding of significantly increased PS products supports the capillary recruitment hypothesis. In 17 of 44 areas, nicotine treatment increased lCBF by 30-150%, K1 of antipyrine by 7-40%, K1 of 3OMG by 5-27%, PS product of antipyrine by 0.20% (mean 7%), and PS product of 3OMG by 0-23% (mean 8%). Nicotine had no effect on blood flow or influx in the remaining 27 areas. The increases in lCBF and K1 of antipyrine were significant, whereas those in K1 of 3OMG and in PS for both antipyrine and 3OMG were not statistically significant. The lack of significant changes in PS products implies that in brain areas where nicotine increased blood flow: (a) essentially no additional capillaries were recruited and (b) blood flow within brain capillary beds rises by elevating linear velocity. The K1 results indicate that the flow increase generated by nicotine will greatly raise the influx and washout rates of highly permeable materials, modestly elevate those of moderately permeable substances, and negligibly change those of solutes with extraction fractions of < 0.2, thereby preserving the barrier function of the blood-brain barrier.

Electronic spin resonance detection of superoxide and hydroxyl radicals during the reductive metabolism of drugs by rat brain preparations and isolated cerebral microvessels.

A spin trapping technique was used to analyze by electron spin resonance (ESR) the formation of oxygen-derived free radicals during the cerebral reductive metabolism of xenobiotics able to undergo a single electron reduction, i.e. quinones, pyridinium compounds and nitroheterocyclics. Paraquat, menadione and nitrofurazone were used as model compounds of these three classes of molecules. ESR spectra indicative of superoxide and hydroxyl radical formation were obtained by incubation of brain homogenates directly within the ESR cavity at 37 degrees C for each of the three molecules tested. These signals were dependent on nucleotide cofactors, and increased in a time-dependent manner. The NADPH and NADH dependent free radical production was further characterized in brain microsomal and mitochondrial fractions, respectively. By using various combinations of reactive species inactivating enzymes (superoxide dismutase, catalase), a metal chelator (deferoxamine), and an hydroxyl trapping agent (dimethylsulfoxide), it was shown that (1) the primary radical generated was the superoxide anion; and (2) a significant production of the hydroxyl radical also occurred, that was secondary to the superoxide anion production. Consistent signals indicative of the production of both oxygen-derived free radicals were obtained when isolated cerebral microvessels which constitute the blood-brain barrier were incubated with the model molecules. This is of particular toxicological relevance, because this barrier represents a key element in the protection of the brain, and is in close contact with blood-born exogenous molecules.

The activity of 1-naphthol-UDP-glucuronosyltransferase in the brain.

Cerebral microsomes catalysed efficiently the glucuronidation of 1-naphthol, this formation of glucuronide being activated by treatment with Triton X-100 or digitonin. Activated microsomes from the brain of the rat conjugated 1-naphthol with an apparent Km of 95 microM and a Vmax of 5.47 nmol/hr mg protein at 30 degrees C. Microsomal uridine diphosphate (UDP)-glucuronosyltransferase activity in brain towards 1-naphthol was not significantly induced by pretreatment of animals with 3-methylcholanthrene or phenobarbital. These data suggest that UDP-glucuronosyltransferases in brain are different from the hepatic enzymes with regard to biochemical parameters and in response to inducers of drug metabolism. The hepatic UDP-glucuronosyltransferase deficiency in Gunn rats was also observed in the brain.

Rapid distribution of intraventricularly administered sucrose into cerebrospinal fluid cisterns via subarachnoid velae in rat.

The intracranial distribution of [14C]sucrose, an extracellular marker infused for 30 s into one lateral ventricle, was determined by autoradiography of frozen-dried brain sections. Within 3.5 min [14C]sucrose appeared in: (i) the third ventricle, including optic, infundibular and mammillary recesses; (ii) the aqueduct of Sylvius; (iii) the velum interpositum, a part of the subarachnoid space that runs along the roof of the third ventricle and contains many blood vessels; (iv) the mesencephalic and fourth ventricles; and (v) the superior medullary velum, a highly vascular extension of the subarachnoid space that terminates at the walls of the mesencephalic and fourth ventricles. Within 5 min, radioactivity was present in the interpeduncular, ambient and quadrigeminal cisterns, which encircle the midbrain. By 10 min, approximately 11% of the radioactivity had passed into the subarachnoid space via a previously undescribed flow pathway that included the velum interpositum and superior medullary velum. At many places along the ventricular system, [14C]sucrose appeared to move from cerebrospinal fluid into the adjacent tissue by simple diffusion, as reported previously (Blasberg R. G. et al. (1974) J. Pharmac. exp. Ther. 195, 73-83; Levin V. A. et al. (1970) Am. J. Physiol. 219, 1528-1533; Patlak C. and Fenstermacher J.D. (1975) Am. J. Physiol. 229, 877-884; Rosenberg G. A. and Kyner W.T. (1980) Brain Res. 193, 56-66; Rosenberg G. A. et al. (1986) Am. J. Physiol 251, F485-F489). Little sucrose was, however, taken up by: (i) circumventricular organs such as the subfornical organ; (ii) medullary and cerebellar tissue next to the lateral recesses; and (iii) the superior and inferior colliculi and cerebral peduncles. For the latter two groups of structures, entry from cerebrospinal fluid was apparently blocked by a thick, multilayered glia limitans. Although [14C]sucrose was virtually absent from the rest of the subarachnoid system after 1 h, it remained in the perivascular spaces and/or walls of pial arteries and arterioles for more than 3 h. Certain transport proteins, protease inhibitors, growth factors and other neurobiologically active materials are present in cerebrospinal fluid, and their distribution to the brain and its blood vessels may be important. The present work shows, in the rat, that the flow of cerebrospinal fluid and the disposition of its constituents is fairly complex and differs among regions. Flow was rapid throughout the ventricular system and into various subarachnoid velae and cisterns, but was surprisingly slow and slight over the cerebral and cerebellar cortices. The cerebrospinal fluid-to-tissue flux of material was relatively free at many interfaces, but was greatly restricted at others, the latter indicating that the old concept of a "cerebrospinal fluid-brain barrier" may hold at such places. Finally, radiolabeled sucrose was retained longer within the walls and perivascular spaces of pial arteries and arterioles than in other subarachnoid tissues; one function of the cerebrospinal fluid system or "third circulation" may thus be delivering factors and agents to these pial blood vessels.

Distribution of cytochrome P450 activities towards alkoxyresorufin derivatives in rat brain regions, subcellular fractions and isolated cerebral microvessels.

The regional and subcellular distributions of rat brain cytochrome P450 and cytochrome P450-dependent activities were examined. Cytochrome P450 was found to be mainly localized in mitochondria in all the six cerebral regions studied. The activities of the isoforms mostly implicated in drug metabolism, cytochromes P450 b and c, were measured by the dealkylation of two alkoxyresorufins, that are sensitive probe substrates for these isoforms. These activities have been measured in microsomal and mitochondrial fractions obtained from six different regions in male rat brains, as well as in microvessels. Resorufin derivatives dealkylation specific activities were higher in brain microsomal fractions than in hepatic ones in all the six regions examined when results were expressed per cytochrome P450 content. These brain microsomal specific activities were also higher than in mitochondrial fractions. Olfactory bulbs showed the highest cytochrome P450 content and activities in both microsomal and mitochondrial fractions. A sex-linked difference in cytochrome P450-dependent activities was also found. After an in vivo inducing pretreatment of rats, only 3-methylcholanthrene induced ethoxyresorufin O-deethylase activity, in the three preparations studied. These results provided (i) direct evidence that cytochromes P450 b and c isoforms are active in brain microsomal fractions, with regional and sex-linked differences, and (ii) the first demonstration of cytochrome P450-dependent activities in isolated rat brain microvessels.

Subcellular localization of cytochrome P450, and activities of several enzymes responsible for drug metabolism in the human brain.

We studied the subcellular distribution of cytochrome P450 and related monooxygenase activities in six regions of human brains removed at autopsy. The content of total cytochrome P450 was found to be at least nine times higher in the mitochondrial fraction than in the microsomes in all the regions studied. However, cytochrome P450-dependent enzymatic activities which are representative of different isoforms metabolizing exogenous molecules exhibited a microsomal prevalence, a situation previously observed in rat brain. The other drug-metabolizing enzymes catalysing functionalization and conjugation reactions, presented the following characteristics in human brain: (i) a low activity of NADPH-cytochrome P450 reductase, which also catalyses the reduction of some xenobiotics; (ii) a high specific activity of the membrane-bound epoxide hydrolase; (iii) among the enzymes catalysing conjugation reactions, 1-naphthol-UDP-glucuronosyltransferase activity was barely or not detectable, whereas the mean glutathione-S-transferase activity was 15 times higher than the activity measured in rat brain. The presence of several drug-metabolizing enzyme activities in human brain microvessels, and particularly the high activity of epoxide hydrolase, suggests a participation of these enzymes in the metabolic blood-brain barrier.

Evidence for drug metabolism as a source of reactive species in the brain.

Several pathways for reactive species formation involving xenobiotic metabolism exist in the brain. They include oxidative activation by different enzymatic systems like cytochrome P-450 and monoamine oxidases, and superoxide radical production issued from reductive xenobiotic metabolism. They may contribute to cellular impairment observed in various physiopathological situations.

Drug metabolizing enzymes in the rat pituitary gland.

Brain protection against chemicals is mainly provided by the specific properties of cerebral microvessels forming the blood-brain barrier. In addition, several drug metabolizing enzymes have been evidenced both in brain tissue and in cerebral capillaries, suggesting their participation in the enzymatic protection of this organ. The pituitary gland, like true circumventricular organs, lacks a tight vascular endothelium and therefore is especially sensitive to blood-native toxic or pharmacologically active molecules. We report here the presence of cytochrome P-450 in the pituitary gland and its main mitochondrial localization. The O-dealkylase activity measured towards 7-benzoxyresorufin, a substrate for the main cytochrome P-450 isoforms involved in the metabolism of xenobiotics, was 5 times higher in the pituitary gland than in the brain cortex. Similarly, microsomal epoxide hydrolase, which inactivates reactive epoxides to trans diol molecules, and two conjugating enzymes, 1-naphthol UDP-glucuronosyltransferase and glutathione-S-transferase, display respectively 6, 4 and 7 times higher activities in the pituitary gland. 7-Benzoxyresorufin-O-dealkylase, 1-naphthol UDP-glucuronosyltransferase and membrane-bound epoxide hydrolase activities were significantly increased in the pituitary gland as an adaptive response to an in vivo treatment by an exogenous inducer, 3-methylcholanthrene. These results suggest that these enzymatic systems play a role in the protection of the pituitary gland towards drugs or toxic substances.

Brain drug delivery, drug metabolism, and multidrug resistance at the choroid plexus.

The choroid plexuses (CPs) have the capability to modulate drug delivery to the cerebrospinal fluid (CSF) and to participate in the overall cerebral biodisposition of drugs. The specific morphological properties of the choroidal epithelium and the existence of a CSF pathway for drug distribution to different targets in the central nervous system suggest that the CP-CSF route is more significant than previously thought for brain drug delivery. In contrast to its role in CSF penetration of drugs, CP is also involved in brain protection in that it has the capacity to clear the CSF from numerous potentially harmful CSF-borne exogenous and endogenous organic compounds into the blood. Furthermore, CP harbors a large panel of drug-metabolizing enzymes as well as transport proteins of the multidrug resistance phenotype, which modulate the cerebral bioavailability of drugs and toxins. The use of an in vitro model of the choroidal epithelium suitable for drug transport studies has allowed the demonstration of the choroidal epithelium acting as an effective metabolic blood-CSF barrier toward some xenobiotics, and that a vectorial, blood-facing efflux of conjugated metabolites occurs at the choroidal epithelium. This efflux involves a specific transporter with characteristics similar to those of the multidrug resistance associated protein (MRP) family members. Indeed, at least one member, MRP1, is largely expressed at the CP epithelium, and localizes at the basolateral membrane. These metabolic and transport features of the choroidal epithelium point out the CP as a major detoxification site within the brain.

Symposium overview: the role of glutathione in neuroprotection and neurotoxicity.

Although the cytoprotective effects of glutathione (GSH) are well established, additional roles for GSH in brain function are being identified that provide a pharmacological basis for the relationship between alterations in GSH homeostasis and the development of certain neurodegenerative processes. Thus, GSH and glutathione disulfide (GSSG) appear to play important functional roles in the central nervous system (CNS). A symposium, focussing on the emerging science of the roles of GSH in the brain, was held at the 37th annual meeting of the Society of Toxicology, with the emphasis on the role of glutathione in neuroprotection and neurotoxicity. Jean Francois Ghersi-Egea opened the symposium by describing the advances in our understanding of the role of the blood-brain and blood-cerebral spinal fluid (CSF) barriers in either limiting or facilitating the access of xenobiotics into the brain. Once within the brain, a multitude of factors will determine whether a chemical causes toxicity and at which sites such toxicity will occur. In this respect, it is becoming increasingly clear that GSH and its various conjugation enzymes are not evenly distributed throughout the brain. Martin Philbert discussed how this regional heterogeneity might provide a potential basis for the theory of differential sensitivity to neurotoxicants, in various regions of the brain. For certain chemicals, GSH provides neuroprotection, and Edward Lock discussed the selective toxicity of 2-chloropropionic acid (CPA) to the cerebellum and how its modification by modulating brain thiol status provides an example of GSH acting in neuroprotection. The sensitivity of the cerebellum to CPA may also be linked to the ability of this compound to activate a sub-type of the NMDA receptor. Thus, GSH and cysteine alone, or perhaps as conjugates with xenobiotics, may play a role in excitotoxicity via NMDA receptor activation. In contrast, certain chemicals may be converted to neurotoxicants following conjugation with GSH, and Arthur Cooper described how the pyridoxal 5'-phosphate-dependent, cysteine conjugate beta-lyases might predispose the brain to chemical injury in a GSH-dependent manner. The theme of GSH as a potential mediator of chemical-induced neurotoxicity was extended by Terrence Monks, who presented evidence for a role for GSH conjugation in (+/-)-3,4- methylenedioxyamphetamine-mediated serotonergic neurotoxicity.