Gérard Siest

Localization of Drug‐Metabolizing Enzyme Activities to Blood‐Brain Interfaces and Circumventricular Organs

Abstract: The brain, with the exception of the choroid plexuses and Circumventricular organs, is partially protected from the invasion of blood‐borne chemicals by the specific morphological properties of the cerebral micro‐vessels, namely, the tight junctions of the blood‐brain barrier. Recently, several enzymes that are primarily involved in hepatic drug metabolism have been shown to exist in the brain, albeit at relatively low specific activities. In the present study, the hypothesis that these enzymes are located primarily at blood‐brain interfaces, where they form an “enzymatic barrier,” is tested. By using microdissection techniques or a gradient‐centrifugation isolation procedure, the activities of seven drug‐metabolizing enzymes in isolated microvessels, choroid plexuses, meningeal membranes, and tissue from three Circumventricular organs (the neural lobe of the hypophysis, pineal gland, and median eminence) were assayed. With two exceptions, the activities of these enzymes were higher in the three Circumventricular organs and cerebral microvessel than in the cortex. Very high membrane‐bound epoxide hydrolase and UDP‐glucuronosyltransferase activities (approaching those in liver) and somewhat high 7‐benzoxyre‐sorufin‐O‐dealkylase and NADPH‐cytochrome P‐450 reductase activities were determined in the choroid plexuses. The pia‐arachnoid membranes, but not the dura matter, displayed drug‐metabolizing enzyme activities, notably that of epoxide hydrolase: The drug‐metabolizing enzymes located at these nonparenchymal sites may function to protect brain tissue from harmful compounds.

Drug metabolizing enzymes in the brain and cerebral microvessels.

Several families of brain parenchyma and microvessel endothelial cell enzymes can metabolize substrates of exogenous origin. This xenobiotic metabolism includes functionalization and conjugation reactions and results in detoxication, but also possibly in the formation of pharmacologically active or neurotoxic products. The brain is partially protected from chemical insults by the physical barrier formed by the cerebral microvasculature of endothelial cells, which prevents the influx of hydrophilic molecules. These cells provide also, as a result of their drug-metabolizing enzyme activities, a metabolic barrier against penetrating lipophilic substances. The involvement of these enzymatic activities in neurotoxic events, probably responsible for neuronal dysfunctioning and/or death, neurodegenerative diseases and normal aging, is discussed.

Increased Levels of Apolipoprotein D in Cerebrospinal Fluid and Hippocampus of Alzheimer's Patients

Abstract: Apolipoprotein D (apoD) is a member of the lipocalin family of proteins. Most members of this family are transporters of small hydrophobic ligands, although in the case of apoD, neither its physiological function(s) nor its putative ligand(s) have been unequivocally identified. In humans, apoD is expressed in several tissues, including the CNS, and its synthesis is greatly increased during regeneration of rat peripheral nerves. As apoD may have an important function in the nervous system and, particularly, in nerve regeneration, we measured immunoreactive apoD levels in the hippocampus and in CSF of patients with either Alzheimers disease (AD) or other neuropathologies. In parallel, we determined the concentrations of apolipoprotein E (apoE), another apolipoprotein also implicated in nerve regeneration and in the etiology of AD. Levels of apoD but not apoE were increased in the hippocampus of AD patients compared with controls. ApoD concentrations, as determined by radioimmunoassay, were significantly increased in the CSF of AD patients (4.23 ± 1.58 µg/ml) and patients with other pathologies (3.29 ± 1.35 µg/ml) compared with those in the CSF of normal subjects (1.15 ± 0.71 µg/ml). Although the differences were smaller than for apoD, the mean apoE concentrations in the CSF of both groups of patients were also significantly higher than those of controls. In AD patients, apoD, but not apoE, levels in CSF and hippocampus increased as a function of inheritance of the ε4 apoE allele. This study therefore demonstrates that increased apoD levels in the hippocampus and in CSF are a marker of neuropathology, including that associated with AD, and are independent of apoE concentrations.

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.

Genetics strongly determines the wall thickness of the left and right carotid arteries

In 76 supposedly healthy families, we investigated the familial resemblance of left and right carotid intima-media thickness (IMT) measured by B-mode ultrasonography and the impact of the common apolipoprotein E (apo E) polymorphism and the insertion/deletion polymorphism of the angiotensin-converting enzyme (ACE). Genetic factors accounted for about 30% of IMT variation. The insertion/deletion ACE polymorphism did not influence carotid IMT, whereas apoE polymorphism explained about 1.5% of only right carotid IMT variability independently of cholesterol levels. The apo ɛ2 and apo ɛ4 alleles were associated with lower right carotid IMT than was the apo ɛ3 allele. We conclude that genetic factors strongly contribute to IMT variability in healthy people and that the apo E polymorphism may be one of these factors.

Subcellular distribution of cytochrome P-450 in the brain

The subcellular distribution of the monooxygenase complexes in the brain was studied by using subcellular fractionation and characterization of these fractions by marker enzymes. Cytochrome P-450 was found to be mainly localized in both synaptic and non-synaptic mitochondria; only a small quantity of enzyme was also found in the microsomal fraction. Peeling off the outer membrane of mitochondria showed that the protein was retained in the inner membrane fraction. A comparative study among some other species confirmed the mitochondrial prevalence of cerebral cytochrome P-450. A partial purification of the rat brain mitochondrial P-450 was obtained.

Blood activity of Cu/Zn superoxide dismutase, glutathione peroxidase and catalase in Alzheimer's disease : a case-control study

Cu/Zn superoxide dismutase (Cu/Zn SOD), glutathione peroxidase (GPx) and catalase, which are the three main enzymes involved in cellular protection against damage due to oxygen-derived free radicals have been assayed in plasma and erythrocytes obtained from subjects with dementia of the Alzheimer type (DAT) and from controls. Blood samples were obtained from 25 patients with DAT and from age-matched subjects without diagnoses of neurological disease (non-DAT), as well as from younger individuals (reference group). Using appropriate statistical procedures, the three enzyme activities measured in blood of the elderly were decreased if compared to the younger reference group. Moreover, a significant increase in erythrocyte Cu/Zn SOD and catalase activities of DAT patients was observed compared to the non-DAT group. These results are discussed taking the free radical theory of aging into consideration.

Comparison of cytochrome P-450 content and activities in liver microsomes of seven animal species, including man

The cytochrome P-450 content found in human livers obtained post mortem was between 0.21-0.42 nmol/mg protein. The kinetic parameters of the mono-oxygenase activities--Km and Vmax--were determined in liver microsomes for N-demethylation (aminopyrine, benzphetamine, ethylmorphine), O-demethylation (4-nitroanisole), O-deethylation (7-ethoxycoumarin) and hydroxylation (benzo[a]pyrene), in an attempt to establish an inter-species comparison between man and the six animal species studied. The four substrates studied (aminopyrine, benzphetamine, ethylmorphine, benzo[a]pyrene) were shown to be less active in humans than the male rat, which is the most commonly used model. However, other animal species, such as the female Sprague-Dawley rat and the pig, are much more similar to man. From a procedural point of view, the optimal substrate concentrations vary from one experimental species to another. Due to the apparent Km observed, for example, the activities of the guinea-pig require a higher substrate concentration.

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.

Cerebrospinal fluid apolipoprotein E level is increased in late-onset Alzheimer's disease

Worldwide evidence has recently shown that the allele epsilon4 of apolipoprotein E (ApoE) is a genetic risk factor for Alzheimers disease (AD) underlining the possible role of apoE in the physiopathology of AD. To evaluate the usefulness of apoE concentration in pathogenesis of AD, we measured the cerebrospinal fluid (CSF) levels of apoE. CSF apoE level was significantly higher in 38 patients with late-onset AD than in 31 control patients and 47 patients suffering from other neurological and related diseases. Higher levels of CSF apoE were also present in a subset of patients with meningoencephalitis, motor neuron disease, and low back pain. The increase of CSF-apoE in AD is in agreement with results from studies that find an increase of mRNA apoE in the brains of AD patients. Compared to other works, these results underline the importance and the difficulties of the selection of the controls. The CSF apoE level seems to be a reflection of neuronal damage and/or an inflammatory reaction that may be common to AD and other neurological and related diseases.