Biomedical subjects

A Minn

Publications and source records attributed to A Minn.

At least 37 records · Page 2Linked to original sources

PubMed1998

Identification of the uridine diphosphate glucuronosyltransferase isoform UGT1A6 in rat brain and in primary cultures of neurons and astrocytes.

The expression of a phenol uridine diphosphate glucuronosyltransferase (UGT) was investigated in rat brain homogenate and in primary cultures of astrocytes and neurons, by means of model substrates (1-naphthol and 4-methylumbelliferone) assays, Western blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) experiments. Glucuronidation of these substances occurred in cerebral cell or brain homogenates, although to different extents. The specific activity was the highest in astrocytes, with values more than 10- and 100-fold those found in neurons or total brain, respectively. Using antibodies able to recognize several rat liver UGT isoforms, only one protein with an apparent molecular mass of 54 kDa was detected in astrocyte and neuron homogenates and brain microsomes. RT-PCR experiments run with primers specifically designed for the rat liver UGT1A6 revealed amplificons of the expected sizes in accordance with the presence of UGT1A6 mRNA. The nucleotide sequence of the 330-base pair product was 100% homologous to that of exon 1 of rat liver isoform UGT1A6. In conclusion, this work allowed us to identify for the first time a constitutive cerebral UGT isoform identical to rat liver UGT1A6, which glucuronidates planar phenolic substances in cultured astrocytes, neurons, and the entire brain.

Animals↗

PubMed1998

Enhanced GFAP expression in astrocytes of transgenic mice expressing the human brain-specific trypsinogen IV.

We recently identified a cDNA encoding a human brain specific trypsinogen (trypsinogen IV). In order to test whether trypsinogen IV is involved in CNS diseases of, or injury response in, mammalian brain, a mouse model was developed in which the human trypsinogen IV was expressed specifically in neurons. Immunocytochemical analysis of the brains of transgenic mice revealed a striking enhancement of glial fibrillar acidic protein (GFAP) expression in astrocytes. This remarkable astrocytic reaction was detected in the brains of mice as young as 2 months and did not diminish in the older animals we tested. However, we did not find gross evidence for neurodegeneration, nor for reactive microglial cells. The long-term survival of these animals should provide a model with which to study the mechanism of nerve-astroglia interactions. In addition, the possible participation of trypsin IV in the metabolism of the Alzheimer precursor protein (APP) was investigated by immunostaining brains from transgenic mice with beta-amyloid (betaA4) antibodies. Immunocytochemical staining of brains from one year old transgenic mice revealed an intense intracellular betaA4-like signal in neurons.

Amyloid beta-Peptides↗

PubMed1998

Drug metabolizing enzyme activities and superoxide formation in primary and immortalized rat brain endothelial cells.

The activities of several enzymes involved in drug metabolism, NADPH-cytochrome P450 reductase, cytochrome P450 isoforms CYP1A and CYP2B, and uridine diphosphate glucuronosyltransferase (UGT) have been measured in primary cultures of rat cerebrovascular endothelial cells and in the immortalized rat brain endothelial cell line RBE4. These drug metabolizing activities were similar in the microsomes prepared from both cell types, even after 20 passages for RBE4 cells. These results were confirmed by Western immunoblotting analysis, using polyclonal antibodies raised against rat liver enzymes. The superoxide production observed during NADPH-cytochrome P450 reductase-dependent monoelectronic reduction of four xenobiotics, menadione, anthraquinone, nitrofurazone and diquat, was also investigated in these cultured cells at confluence. The rates of radical production were concentration-dependent. The superoxide formation induced by quinone metabolism was comparable in both cell cultures, and high amounts of superoxide radicals were produced even after 20 passages of RBE4 cells. On the other hand, nitrofurazone and diquat metabolism produced weak amounts of superoxide radicals in both cell types. Taken together, these results suggest that RBE4 cell line seems to constitute a valuable in vitro model for studies on the activity of some enzymatic systems involved in drug metabolism at the blood-brain barrier and the functional consequences of their activity.

Animals↗

PubMed1996

The superoxide production mediated by the redox cycling of xenobiotics in rat brain microsomes is dependent on their reduction potential.

Several exogenous molecules undergo enzymatic one-electron reduction leading to radicals which can rapidly react with molecular oxygen to form superoxide anions. We have previously shown that under aerobic conditions a significant superoxide anion production occurred during the NADPH-dependent one-electron reduction of some drugs and xenobiotics by rat brain preparations. We report here for several compounds a fairly good correlation between the reduction potentials (Epc vs. SCE) which ranged between - 230 and - 700 mV in aqueous medium (pH 7.4) or between -700 mV and -1100 mV in the aprotic solvent N,N-dimethylformamide, and the rate of superoxide anion production during their metabolism by rat brain microsomes. The data obtained suggest that the redox potential of most of the molecules assayed was related to their ability to undergo one-electron reduction mediated by flavoenzymes in the rat brain. The main range of reduction potentials corresponding to a large superoxide anion production suggests that the redox cycling of these chemicals was mediated by NADPH-cytochrome P-450 reductase. Therefore the measurement of reduction potentials of drugs and xenobiotics able to reach the brain, and chemically related to quinones, nitroaromatics, nitroheterocyclics and iminiums, may provide information both on their electron affinity and the possibility of one-electron transfer in vivo, and thus on their possible neurotoxicity due to the production of oxygenated free radicals.

Animals↗

PubMed1996

Effects of MPTP on lever-pressing for light extinction in rats.

Rats were daily treated for seven days with 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP) at a dose of 20 mg/kg/day, i.p. Seven days after treatment withdrawal, the rats were individually tested in a brightly lit apparatus containing two levers: an active lever allowing periods of darkness, and an inactive one. The test was performed over two consecutive days, in 20-min sessions. While control rats had a higher number of total active lever pressings than inactive lever pressings, this was not the case for MPTP-treated rats. Control rats decreased their useless active lever pressings and inactive lever pressings across the two sessions, but MPTP-treated rats did not do either. The absence of the differential effect in rats injected with MPTP may be due to a reduction in reinforcement mechanisms caused by the mild depletion of dopamine in the striatum.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed1995

Dextromethorphan O-demethylase activity in rat brain microsomes.

CYP2D, a genetically variable isoform of cytochrome P450, has been characterized mainly in the liver and the brain of mammals by measurement of debrisoquine hydroxylase activity. Moreover, 'poor debrisoquine metabolizer' phenotype is significantly increased in Parkinson's disease patients. We present here the first demonstration that the activity of the CYP2D isoform can be characterized in rat brain microsomes by the measurement of dextromethorphan O-demethylase capacity. The cerebral formation of dextrorphan, an antagonist of the N-methyl-D-aspartate receptor, was inhibited by the presence of quinidine and N-methyl-4-phenylpyridinium (MPP+), a dopaminergic neurotoxin inducing a chemical parkinsonism in humans.

Animals↗

PubMed1994

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.

Animals↗

PubMed1994

Superoxide anion production during monoelectronic reduction of xenobiotics by preparations of rat brain cortex, microvessels, and choroid plexus.

Brain microsomes may produce reactive metabolites during the reductive metabolism of some xenobiotics including drugs. These reactive species can, in turn, react with molecular oxygen to form superoxide radicals (O2.-). We measured the rates of superoxide production by homogenates obtained from three cerebral structures, cortex plus cerebellum, choroid plexus, and microvessels. The molecules assayed were related to quinone, nitroheterocycle, and iminium chemical families. The results we obtained showed a significant correlation between the rate of superoxide anion production and the apparent kinetic parameters (log Km/Vmax) of NADPH-cytochrome P450 reductase activity for these molecules, suggesting the involvement of this enzyme in xenobiotic-induced superoxide production.

Animals↗

PubMed1994

Localization of drug-metabolizing enzyme activities to blood-brain interfaces and circumventricular organs.

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-benzoxyresorufin-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.

Animals↗

PubMed1993

Cloning of the cDNA encoding human brain trypsinogen and characterization of its product.

We designed degenerated oligodeoxyribonucleotide primers derived from amino acid (aa) sequences of the highly conserved active sites of mammalian serine proteases (SPs). These primers were used to selectively amplify, in polymerase chain reactions (PCRs), cDNA fragments coding for a SP. We used poly(A)+RNA from human brain to obtain cDNA fragments and amplified one cDNA encoding a novel SP. The full-length nucleotide (nt) sequence was identified by PCR and screening a genomic library in order to obtain the 5'-region. The deduced as sequence shows a high degree of homology to trypsinogens, except for the first exon. In addition to this brain-specific trypsinogen, there exists a variant of the cDNA in pancreas, differing only in the nt sequence of the first exon. An active form of the trypsin was synthesized in vitro and purified by affinity chromatography using soybean trypsin inhibitor (STI) agarose to demonstrate the trypsin-specific interaction with a naturally occurring inhibitor of trypsins.

Amino Acid Sequence↗

PubMed1993

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.

Animals↗

PubMed1993

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.

Aged↗

PubMed1992

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.

Animals↗

PubMed1991

Enzyme mediated superoxide radical formation initiated by exogenous molecules in rat brain preparations.

The ability of brain tissue preparation to generate superoxide from xenobiotic interactions has been investigated. We showed that a significant superoxide production occurred with different molecules known to undergo a single electron reductive pathway of metabolism, both in a homogenate derived from neuronal and glial cells and in isolated cerebral microvessels which form the blood-brain barrier. Determination of the nucleotide cofactors requirement and data obtained with different subcellular fractions indicated that this production was largely associated with the microsomal fraction in a NADPH-dependent pathway and was probably mediated by NADPH-cytochrome P450 (c) reductase. A significant xenobiotic-mediated production of superoxide also occurred in mitochondria under in vitro conditions. Thus the evidence of reductive pathways of xenobiotic metabolism and the generation of oxygenated free radicals observed are of neurotoxicological significance.

Animals↗

PubMed1991

In vivo study of the elimination from rat brain of an intracerebrally formed xenobiotic metabolite, 1-naphthyl-beta-D-glucuronide.

Among the drug-metabolizing enzymes present in the rat brain, one form of UDP-glucuronyltransferase catalyzes the formation of the polar metabolite 1-naphthyl-beta-D-glucuronide from 1-naphthol. We measured the activity of this isoform in different brain regions and showed its heterogeneous distribution. Conjugation activities were found to be the highest in the olfactory bulbs (25.4 nmol/h/mg protein) and lowest in the cerebellum (4.5 nmol/h/mg protein). As the blood-brain barrier prevents the passage of hydrosoluble molecules, we studied in vivo the characteristics of the efflux of labeled 1-naphthyl-beta-D-glucuronide injected into the lateral ventricle and the cortex tissue, using tritiated water and labeled inulin as reference compounds. The results reported here indicate that intracerebrally formed glucuronide is cleared from brain tissue by both diffusion and a saturable efflux process.

Animals↗

PubMed1991

Identification of novel phencyclidine metabolites formed in vitro by rabbit microsomal metabolism.

1. Phencyclidine (PCP) was incubated with rabbit liver and brain microsomal fractions, and the structures of metabolites formed by oxidation determined by g.l.c.-mass spectrometry. 2. The formation of several known mono- and di-hydroxylated metabolites, as well as two new metabolites, was seen in the liver preparations. 3. Hydroxylated PCP metabolites were also formed after incubation of PCP with brain microsomes, indicating that PCP biotransformation may occur in the brain itself.

Animals↗

PubMed1990

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.

Animals↗

PubMed1990

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.

Aged↗