[Astrocytic cytochromes p450: an enzyme subfamily critical for brain metabolism and neuroprotection]. / Les cytochromes P450 astrocytaires, une famille d'enzymes clés dans le métabolisme cérébral et la neuroprotection.

INTRODUCTION: Astrocytes are involved in multiple brain functions in physiological conditions. Cytochromes P450 are expressed in astrocytes and play a role in brain metabolism and in neuroprotection. BACKGROUND: Although the levels of various cytochromes P450 in brain regions are low, these enzymes were reported to be expressed at relatively high level in astroglial cells and may play a critical role in the biotransformation of endogenous or exogenous compounds. Astroglial cytochromes P450 expression suggests a putative capacity to metabolize psychoactive or lipophilic xenobiotics in situ, associated with pharmacological and/or toxicological consequences. Astrocytes appear to be the most active steroidogenic cells in the brain, expressing neurosteroïdogenic cytochromes P450 and producing various neurosteroids. Cytochrome P450 epoxygenase enzymes, which catalyze the formation of vasoactive compounds are also present in astrocytes, contributing to the regulation of the cerebral blood flow. PERSPECTIVES AND CONCLUSION: This review underlines the crucial roles of astroglial cytochromes P450 in brain functions. Identification of the molecular mechanisms involved in the regulation of these enzymes could open therapeutic perspectives and improve our understanding in neuroprotection.

Serum myeloperoxidase concentration in a healthy population: biological variations, familial resemblance and new genetic polymorphisms.

Myeloperoxidase (MPO) has been involved in the pathogenesis of several diseases through excessive production of reactive oxygen species (ROS) as well as through its genetic polymorphism. The aims of this study were to identify the factors affecting MPO serum concentration, to study the familial resemblance of MPO levels and to investigate the association between newly described MPO polymorphisms as well as the G-463A one and MPO levels in a healthy population. MPO serum concentrations were measured by an enzymatic immuno-assay (EIA) in 82 healthy families of the STANISLAS Cohort and MPO genotype, determination was performed using PCR-restriction fragment length polymorphism or allele specific oligonucleotide assay. MPO concentrations were significantly higher in parents than in offspring. The factors affecting MPO levels were age, the number of white cells, smoking in fathers and oral contraceptive intake in mothers. They explain from 12.4% up to 35.9% of MPO variability in men and women, respectively. Family correlations of MPO concentrations were of similar magnitude. The -129A allele of a newly described G-129A substitution was significantly associated with decreased MPO levels, whereas the -463A allele was suggested to be associated with increased levels of lipid variables. In this study, we identified factors affecting MPO serum concentrations and showed that molecular variations of the gene have only a weak influence on MPO variability. In contrast, the association between the G-463A polymorphism and lipid levels would suggest a possible implication of MPO in the risk of cardiovascular diseases. These results have to be confirmed and further investigations will be conducted in that way.

Effect of apolipoprotein E on cell viability in a human neuroblastoma cell line: influence of oxidation and lipid-association.

Apolipoprotein E (apoE) whose polymorphic expression is widely associated with Alzheimer's disease (AD) is one of the most studied protein present in cerebral amyloid deposits. Native or fragments of apoE are known to exert neurotoxic effects. We evaluated the effects of apoE oxidation and lipid-association on the viability of human neuroblastoma IMR32 cells. We show that apoE affects cell viability only when it is lipid-associated and applied at a concentration near to that found in plasma, and this whatever the isoform. Oxidized phospholipid-associated apoE has a similar impact on cell viability. These findings show the necessity of including apoE into phospholipids when studying its effect on cell metabolism and underline the probable intervention of surface heparan sulfate proteoglycans (HSPG). It also warrants further studies in order to delineate the pathophysiological importance of apoE.

Differential oxidation of apolipoprotein E isoforms and interaction with phospholipids.

Accumulation of oxidized proteins has been demonstrated in the brain of patients suffering from Alzheimer's disease (AD). Among the proteins found in cerebral amyloid deposits, apolipoprotein (apo) E is a polymorphic protein which one specific isoform, apo E4, has been widely associated with AD. Apo E may be linked with AD by its isoform-specific interaction with lipids or other proteins in amyloid plaques. Using the myeloperoxidase oxidative system, we report that oxidation of the three recombinant apo E isoforms is differential (as estimated using immunoblot and high-performance liquid chromatography analysis), with apo E4 being more susceptible than apo E3, which in turn is much more susceptible than apo E2. In addition, susceptibility to thrombin proteolysis is reduced when apo E is oxidized, and oxidation of apo E decreases its incorporation into phospholipid discs by approximately 50%. Oxidation of apo E may contribute to inefficient lipid recycling in the brain, particularly regarding apo E4 and E3. Our results link and strengthen both the E4 allele linkage with AD and the role of protein oxidation in AD. The cerebral mechanisms underlying apo E oxidation and/or myeloperoxidase functions in vivo remain to be assessed.

Apolipoprotein E is highly susceptible to oxidation by myeloperoxidase, an enzyme present in the brain.

Apolipoprotein E, the most common apolipoprotein found in the brain, is linked to several pathologies like Alzheimer's disease. Apolipoprotein E directly binds to beta-amyloid with a strong affinity. Myeloperoxidase, a protein secreted by neutrophils and involved in the inflammatory process, is also present in the brain. In vitro myeloperoxidase oxidation of recombinant human apolipoprotein E leads to fragmentation of the protein with low concentrations of hydrogen peroxide and polymerization with higher concentrations. Comparison with bovine serum albumin shows a higher susceptibility of apolipoprotein E to myeloperoxidase oxidation, which may have importance in the Alzheimer's disease process.

The rat, a useful animal model for pharmacological studies on apolipoprotein E.

Apolipoprotein E is a major protein component of lipoproteins and plays an important role in cholesterol transport. The structure of the gene and the polymorphism of apolipoprotein E have been studied in human and rat, which show similar structures of apolipoprotein E. The wide tissue distribution of this apolipoprotein suggests diverse functions like cholesterol distribution between cells, intracellular cholesterol trafficking and tissue reparation. Nevertheless, the presence of apolipoprotein E in atherosclerotic plaques and amyloid deposits in brains of Alzheimer's disease patients also indicate pathologic functions staying misunderstood. The aim of this paper is to review the present knowledge on the distribution of apolipoprotein E between the different organs with the related functions and to make an overview of the implications of this apolipoprotein is physiological events and pathological states in the rat. The rat is widely used for drug metabolism studies. Its serum levels are 5-10 times higher than in human and thus this animal provides an useful pharmacological model to elucidate the functions of apo E.

Blood-brain interfaces: relevance to cerebral drug metabolism.

The brain, with the exception of the circumventricular organs (CVOs), is partially protected from the invasion of blood-borne chemicals by the tight junctions that link adjacent cerebral endothelial cells and form the structural basis of the blood-brain barrier (BBB). In addition to the BBB, the epithelial layer of the choroid plexuses and the barrier layer of the arachnoid membrane complex comprise a second system for protecting the brain, a system often referred to as the blood-cerebrospinal fluid (CSF) barrier. In the past several years, several enzymes that are involved in hepatic drug metabolism have been found in the small microvessels from brain, the choroid plexuses, and the leptomeninges (pia plus arachnoid mater) as well as in some CVOs. These drug-metabolizing systems are inducible and may act at these various interfaces as 'enzymatic barriers' to influx. In particular, the activities of these enzymes in choroidal tissue are so high that the choroid plexuses can well be the major site of drug metabolism in the brain. The fate of intracerebrally formed polar metabolites and the potential of the blood-brain and blood-CSF barriers as sites for metabolic activation-induced neurotoxicity are discussed.

Apolipoprotein E: an important gene and protein to follow in laboratory medicine.

The human apolipoprotein (apo) E gene is polymorphic, with three common alleles (epsilon 2, epsilon 3, epsilon 4) coding for three isoforms (E2, E3, E4). The isoforms differ from each other by a single amino acid substitution, and also differ in their binding affinity for the four apo E receptors. Apo E polymorphism is an important determinant of risk for the development of cardiovascular and Alzheimer diseases, the prevalence of the epsilon 4 allele being increased in both kinds of patients compared with control subjects. Furthermore, the prevalence of the epsilon 4 allele differs among populations (range 5-40%, respectively, for Taiwanese and Papua New Guineans). Genotyping or phenotyping needs to be introduced in clinical laboratories. The choice of the method should be based on the types of patients who are examined. The apo E genotype is also a determinant of apo E plasma concentration. Standardization of apo E measurement is an important prerequisite before investigating the clinical interest of plasma apo E concentration. Determination of apo E genotype/phenotype and later the plasma concentration are expected to yield useful clinical laboratory information.

[Drug conjugation in the brain]. / La conjugaison des médicaments dans le cerveau.

Several drugs and environmental pollutants can cross the blood-brain barrier and reach the brain. This organ possesses an enzymatic equipment able to metabolize xenobiotics, therefore facilitating their elimination and maintaining brain homeostasis. This metabolism may result in the formation of toxic metabolites, which endanger neuronal integrity and may result in neurological disturbances. The main function of the conjugation enzymes present in the brain parenchyma and blood-brain or blood-cerebrospinal fluid interfaces is the detoxification of these reactive molecules, thus protecting the central nervous system. This review presents the main cerebral conjugating enzymes, and shows the interest for the design of new drugs of the knowledge of their activities.

Induction and immunological characterization of the uridine diphosphate-glucuronosyltransferase conjugating 1-naphthol in the rat choroid plexus.

The uridine diphosphate-glucuronosyltransferase isoform conjugating 1-naphthol has a very high activity in the rat choroid plexus. We showed that antibodies raised against the main liver isoenzyme cross-reacted with a choroidal protein exhibiting the same molecular weight as the hepatic form. Both enzymes had a similar affinity for 1-naphthol. After an in vivo treatment by 3-methylcholanthrene, a 3-fold increase of the activity in both the liver and choroid plexuses was observed. These results suggest that the choroidal and hepatic enzymes conjugating 1-naphthol are identical. This high metabolic activity suggests a metabolic protection of the brain by the choroid plexus.

Uridine diphosphate-glucuronosyltransferase activities in rat brain microsomes.

The glucuronidation capacity of rat brain microsomes was investigated using a series of chemically related phenolic compounds and fatty acids which are usually glucuronidated in vivo. Most of the phenols assayed were glucuronidated, but no glucuronide formation was detected for stearic and alpha-linolenic acids, 4-methylphenol, bilirubin, morphine, dopamine and serotonin. The activity of uridine diphosphate-glucuronosyltransferase (UGT) towards 1-naphthol represented 0.28% of that obtained with liver microsomes. The inhibitory effects on the formation of 1-naphthol glucuronide of some endogenous and exogenous substances were investigated. The results suggest that only the isoform of UGT conjugating 1-naphthol is present in rat brain.

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.

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.