Alain Minn

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.

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.

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.

TRANSENDOTHELIAL PERMEABILITY CHANGES INDUCED BY FREE RADICALS IN AN IN VITRO MODEL OF THE BLOOD-BRAIN BARRIER

In the present study, we investigated the changes in blood-brain barrier (BBB) permeability following brain endothelial cell exposure to different xenobiotics able to promote free radical generation during their metabolism. Our in vitro BBB model consisted of confluent monolayers of immortalized rat brain capillary endothelial cells (RBE4) grown on collagen-coated filters in the presence of C6 glioma cells grown in the lower compartment. We have recently shown that a range of xenobiotics, including menadione, nitrofurazone, and methylviologen (paraquat) may undergo monoelectronic redox cycling in isolated brain capillaries, giving rise to reactive oxygen species. In this study, addition of 100 microM menadione to the culture medium for 30 min significantly increased the permeability of endothelial cell monolayers to radiolabeled sucrose. The effect on endothelial permeability induced by menadione was dose-dependent and reversible. These permeability changes preceded the onset of cell death, as assessed by the Trypan blue exclusion method. Pre-incubation with superoxide dismutase and catalase blocked changes in sucrose permeability to control levels in a dose-dependent manner, suggesting the involvement of reactive oxygen species in menadione-induced BBB opening.

Drug Transport into the Mammalian Brain: The Nasal Pathway and its Specific Metabolic Barrier

It is generally accepted that the rate of entry into and distribution of drugs and other xenobiotics within the central nervous system (CNS) depends on the particular anatomy of the brain microvessels forming the blood-brain barrier (BBB), and of the choroid plexus forming the blood-cerebrospinal fluid barrier (CSF), which possess tight junctions preventing the passage of most polar substances. Drug entry to the CNS also depends on the physicochemical properties of the substances, which can be metabolised during this transport to pharmacologically inactive, non-penetrating polar products. Finally, the entry of drugs may be prevented by multiple complex specialized carriers, which are able to catalyse the active transport of numerous drugs and xenobiotics out of the CNS. Nasal delivery is currently considered as an efficient tool for systemic administration of drugs that are poorly absorbed via the oral route, and increasing evidence suggests that numerous drugs and potentially toxic xenobiotics can reach the CNS by this route. This short review summarizes recent knowledge on factors controlling the nasal pathway, focusing on drug metabolising enzymes in olfactory mucosa, olfactory bulb and brain, which should constitute a CNS metabolic barrier.

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.

Pefloxacin-Induced Achilles Tendon Toxicity in Rodents: Biochemical Changes in Proteoglycan Synthesis and Oxidative Damage to Collagen

ABSTRACT Despite a relatively low incidence of serious side effects, fluoroquinolones and the fluoroquinolone pefloxacin have been reported to occasionally promote tendinopathy that might result in the complication of spontaneous rupture of tendons. In the present study, we investigated in rodents the intrinsic deleterious effect of pefloxacin (400 mg/kg of body weight) on Achilles tendon proteoglycans and collagen. Proteoglycan synthesis was determined by measurement of in vivo and ex vivo radiosulfate incorporation in mice. Collagen oxidative modifications were measured by carbonyl derivative detection by Western blotting. An experimental model of tendinous ischemia (2 h) and reperfusion (3 days) was achieved in rats. Biphasic changes in proteoglycan synthesis were observed after a single administration of pefloxacin, consisting of an early inhibition followed by a repair-like phase. The depletion phase was accompanied by a marked decrease in the endogenous serum sulfate level and a concomitant increase in the level of sulfate excretion in urine. Studies of ex vivo proteoglycan synthesis confirmed the in vivo results that were obtained. The decrease in proteoglycan anabolism seemed to be a direct effect of pefloxacin on tissue metabolism rather than a consequence of the low concentration of sulfate. Pefloxacin treatment for several days induced oxidative damage of type I collagen, with the alterations being identical to those observed in the experimental tendinous ischemia and reperfusion model. Oxidative damage was prevented by coadministration of N-acetylcysteine (150 mg/kg) to the mice. These results provide the first experimental evidence of a pefloxacin-induced oxidative stress in the Achilles tendon that altered proteoglycan anabolism and oxidized collagen.

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.

Evidence for neurogenic transmission inducing degenerative cartilage damage distant from local inflammation.

OBJECTIVE To investigate involvement of the nervous system in ipsilateral and contralateral joint inflammation. METHODS Freunds complete adjuvant (CFA; 1 mg or 1 microg) was injected unilaterally and the messages (a) from the hind paw to the ipsilateral and contralateral knees and (b) from one knee to the contralateral knee were analyzed. The degenerative impact of the local injury on distant cartilage was assessed using patellar proteoglycan synthesis as an indicator. Neurogenic mechanisms were blocked either by spinal cord compression or by injection of neurokinin 1 (NK-1) antagonist, or they were mimicked by intraarticular injection of substance P. The data were compared with those gathered in a model of systemic inflammation, characterized by fever and serum interleukin-6 production. RESULTS After unilateral subcutaneous injection of CFA, proteoglycan anabolism decreased bilaterally. Spinal cord compression and administration of the NK-1 antagonist inhibited the response in the contralateral limb. Following 1 mg CFA subcutaneous injection, the ipsilateral response implicated both neurogenic and systemic mechanisms, whereas the nervous system alone was implicated after 1 microg subcutaneous CFA injection. The 1 microg CFA intraarticular injection induced a degenerative contralateral signal, which was abolished by spinal cord compression and by pretreatment with the NK-1 antagonist. Intraarticular injection of 1 microg CFA also induced an ipsilateral increase of anabolism, which was enhanced by spinal cord compression. Similar results were obtained after intraarticular injections of substance P. These effects were not reproduced with turpentine treatment, a systemic model, in which spinal cord compression had no effect. CONCLUSION A unilateral inflammation can induce, by neurogenic mechanisms, distal bilateral degeneration of articular cartilage, implicating involvement of neuropeptides.