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B Clement

Publications and source records attributed to B Clement.

At least 37 records · Page 2Linked to original sources
PubMed1997

New NO-donors with antithrombotic and vasodilating activities, Part 17. Arylazoamidoximes and 3-arylazo-1,2,4-oxadiazol-5-ones.

Seven arylazoamidoximes (3), six phenoxycarbonyl derivatives (4), and six 1,2,4-oxadiazol-5-ones (5) have been prepared and their structure and purity established by spectroscopy and elemental analysis. In the EI mass spectra ready elimination of NO from the title amidoximes was observed. A new addition reaction of 3a with hydrochloric acid to 4-chlorophenylhydro-azoamidoxime 7 is described. The compounds were tested for nitric oxide dependent biological properties, i.e. platelet aggregation, antithrombotic effects, and decrease in blood pressure. In arterioles of rats 5/19 compounds inhibited the formation of thrombi with a laser beam by > or = 20% 2 h after oral administration of 60 mg/kg. Among these are three amidoximes (3a, 3e, 3f), one phenoxycarbonyl derivative (4a), and one oxadiazolone (5a). With the 4-chlorophenylazoamidoxime 3c a long lasting (24 h) decrease of blood pressure in spontaneously hypertensive rats was observed. Microsomal fractions of rat liver oxidize arylazoamidoximes and generate nitric oxide (e.g. 3a and 3b). NO was measured by the oxyhemoglobin assay. The influence of SOD, pretreatment of the rats with dexamethasone, as well as kinetic parameters were determined. Type 3 compounds, therefore, are a new class of NO donors. Type 4 and 5 compounds function as their prodrugs.

Animals↗
PubMed1997

Formation of guanoxabenz from guanabenz in human liver. A new metabolic marker for CYP1A2.

The in vitro N-hydroxylation of guanabenz as well as the corresponding N-dehydroxylation of guanoxabenz has been previously detected in biotransformation studies with microsomal fractions of different species including human hepatic microsomes. Furthermore, the N-hydroxylation of guanabenz was found to be catalyzed by enriched cytochrome P450 (P450) fractions in reconstituted systems. Strong correlations between 7-ethoxyresorufin O-deethylation (r = 0. 96; p < 0.001), caffeine N-demethylation (r = 0.92; p < 0.001), respectively, and guanabenz N-hydroxylation activities were demonstrated in 10 human liver microsomal preparations. Studies with microsomes from human B-lymphoblastoid cell lines expressing human cytochrome P450 enzymes proved that CYP1A2 is the major isozyme responsible for this metabolic pathway. Further, P450 isozymes did not show any detectable conversion rates. The reaction was inhibited in presence of the potent CYP1A2 inhibitors alpha-naphthoflavone (7, 8-benzoflavone) and furafylline. The N-reduction of guanoxabenz to guanabenz exhibits a significant correlation to the benzamidoxime N-reduction after incubation with 10 human liver microsomal preparations (r = 0.97; p < 0.001). The formation of benzamidine from benzamidoxime was described previously to be catalyzed by the benzamidoxime reductase. These results suggest that the guanabenz N-hydroxylation is mediated via CYP1A2, whereas the corresponding guanoxabenz N-reduction is catalyzed by an enzyme system composed of cytochrome b5, NADH cytochrome b5-reductase, and benzamidoxime reductase. The high affinity of guanabenz to CYP1A2 and the distinct selectivity of this P450 isozyme toward guanabenz confirms the in vitro guanabenz N-hydroxylation to be a suitable metabolic marker for CYP1A2 in biotransformation studies.

Antihypertensive Agents↗
PubMed1996

Microsomal catalyzed N-hydroxylation of guanabenz and reduction of the N-hydroxylated metabolite: characterization of the two reactions and genotoxic potential of guanoxabenz.

The N-reduction of the centrally acting alpha 2-adrenoreceptor agonist guanoxabenz (Benzérial), an N-hydroxyamidinohydrazone, to the amidinohydrazone guanabenz (Wytensin, Hipten, Rexitene) by microsomal fractions from rabbits, pigs and humans has been detected in vitro. The conversion rates with rabbit microsomal fractions were markedly slower than those in the cases of fractions from humans and pigs. It was also possible to demonstrate the N-oxidation of guanabenz to guanoxabenz by the use of microsomal fractions from rabbits, pigs, and humans. Furthermore, the oxidation was also observed in reconstituted systems in the presence of enriched cytochrome P450 fractions, purified isoenzyme P450 2C3, and heterologously expressed P450 2C3 of the subforms 6 beta H and 6 beta L. The analyses were performed with a newly developed HPLC technique and were confirmed by LC-MS methods. The kinetic parameters determined for the metabolic cycle (bioreversible reactions) are indicative of a predominance of the reduction of guanoxabenz to guanabenz in vivo. Accordingly, guanoxabenz in part constitutes a prodrug of guanabenz. Examination of guanabenz and guanoxabenz for mutagenicity by means of the Ames test revealed that guanoxabenz has pronounced mutagenic effects in the strains TA 98 and TA 1537. Guanabenz did not exhibit mutagenicity so that the N-reduction of guanoxabenz has significance in terms of detoxification.

Adrenergic alpha-Agonists↗
PubMed1996

HIP/PAP is an adhesive protein expressed in hepatocarcinoma, normal Paneth, and pancreatic cells.

Human hepatocarcinoma-intestine-pancreas (HIP) cDNA, isolated from a hepatocellular carcinoma, encodes a C-type lectin. According to published cDNA sequences, HIP protein is identical to human pancreatitis-associated protein (PAP). In these sequences, a putative signal peptide and the carbohydrate recognition domain (CRD) can be recognized. In the present study, we established transgenic mice to drive the production of soluble recombinant HIP/PAP protein in the milk of lactating animals; using this model, we showed that HIP/PAP protein was secreted after suitable cleavage of the potential signal peptide. Moreover, we also produced HIP/PAP protein by Escherichia coli cultures performed to generate specific antibodies. These antibodies enabled the detection of HIP/PAP protein in normal intestine and pancreas (both in endocrine and exocrine cells), e.g., intestinal neuroendocrine and Paneth cells, pancreatic islets of Langerhans, and acinar cells. HIP/PAP protein was also identified in the cytoplasm of tumoral hepatocytes but not in nontumoral hepatocytes. Finally, HIP/PAP protein activity was tested and we showed that HIP/PAP induced the adhesion of rat hepatocytes and bound strongly to extracellular matrix proteins (laminin-1, fibronectin), less strongly to type I and IV collagen, and not at all to heparan sulfate proteoglycan. In conclusion, these results showed that HIP/PAP protein was matured on secretion. We also demonstrated that HIP/PAP protein was specifically expressed in hepatocarcinoma cells and interacted with rat hepatocytes and the extracellular matrix. Taken overall, these results suggest that HIP/PAP protein may be of potential importance to liver cell differentiation/proliferation.

Acute-Phase Proteins↗
PubMed1996

[Biotransformation in hen's eggs. Metabolic transformation of p-nitrophenol].

Not only phase I reactions but also phase II reactions can be detected using embryonated chicken eggs as an alternative method for biotransformation studies. Previously reported investigations which enabled the detection of the phase II products of 7-ethoxycoumarin in the allantois after enzymatic conjugate cleavage have since been supplemented by the development of a direct method. The glucuronidation and sulfation of p-nitrophenol were chosen as model reactions for this direct detection of conjugation reactions. The utility of the previously developed method for application to biotransformation investigations involving the detection of metabolites in the allantois in fertile chicken eggs has now been substantiated for the model substance p-nitrophenol. p-Nitrophenyl sulfate and p-nitrophenyl glucuronide were directly identified as phase II metabolites after incubation and work-up by HPLC analysis as well as comparison of their spectra with those of authentic substances. The conversion rates obtained are very high and well comparable with those of in vivo investigations in chickens. It would seem that the metabolism of hydrophilic substances can be advantageously studied in chicken eggs.

Animals↗
PubMed1995

In vivo spin trapping of glyceryl trinitrate-derived nitric oxide in rabbit blood vessels and organs.

BACKGROUND: The objectives of this study were (1) to assess glyceryl trinitrate (GTN)-derived nitric oxide (NO) formation in vascular tissues and organs of anesthetized rabbits in vivo, (2) to establish a correlation between tissue NO levels and a biological response, and (3) to verify biotransformation of GTN to NO by cytochrome P-450. METHODS AND RESULTS: NO was trapped in tissues in vivo as a stable paramagnetic mononitrosyl-iron-diethyldithiocarbamate complex [NOFe(DETC)2]. After removal of the tissues, NO was determined by cryogenic electron spin resonance spectroscopy. NO formation in vitro was assessed by spin trapping and by activation of soluble guanylyl cyclase. The GTN-elicited decrease in coronary perfusion pressure was monitored in isolated, constant-flow perfused rabbit hearts. NO was not detected in control tissues. In GTN-treated rabbits, NO formation was higher in organs than in vascular tissues and higher in venous than in arterial vessels. In isolated hearts, ventricular NO levels and decreases in coronary perfusion pressure achieved by GTN were closely correlated. Purified cytochrome P-450 catalyzed NO formation from GTN in a P-450-NADPH reductase- and NADPH-dependent fashion. CONCLUSIONS: Since GTN-derived NO formation in myocardial tissue correlates to the GTN-elicited vasodilator response, we conclude that GTN-derived NO detected in vivo correlates with the systemic effects of GTN. Therefore, the higher rate of NO formation detected in veins compared with arteries explains the preferential venodilator activity of GTN. High NO formation in cytochrome P-450-rich organs in vivo and efficient NO formation from GTN by cytochrome P-450 in vitro highlights the importance of this pathway for NO formation from GTN in the intact organism.

Animals↗
PubMed1995

On the mechanism of nitric oxide formation upon oxidative cleavage of C = N(OH) bonds by NO-synthases and cytochromes P450.

Microsomal liver cytochromes P450 catalyze the oxidative cleavage of the C = NOH bond of many ketoximes, amidoximes and guanidoximes, and NO synthases catalyze the oxidation of N omega-hydroxy-L-arginine to citrulline and NO. All these oxidations appear to be performed either by the FE(II) O2 complex of these hemoproteins or by O2.- which is formed by its decomposition. This leads to a unifying view of the mechanisms of P450- and NOS-dependent oxidative cleavage of C = NOH bonds, the relative contribution of Fe(II) O2.- being very different in NO-synthase and cytochromes P450.

Amino Acid Sequence↗
PubMed1995

N-hydroxylation and N-dealkylation by P4502C3 of N-methylbenzamidine: N-oxygenation and N-oxidative dealkylation of one functional group.

1. The first detection of a microsomal N-hydroxylation of an N-alkylated benzamidine possessing alpha-H atoms by P450 is now reported in the present in vitro biotransformation studies. 2. The newly found metabolites, N-hydroxy-N-methylbenzamidine and N-methylbenzamidoxime, were identified after hplc separation by comparison of their retention times with those of synthetic reference compounds and by comixing methods. N-hydroxy-N-methylbenzamidine exists predominantly in the aminonitrone form and constitutes a novel type of metabolite. 3. By means of reconstitution experiments with purified P4502C3 from rabbit liver and with purified variants of 2C3 expressed in Escherichia coli, it has been shown that the N-hydroxylation and the N-dealkylation of N-methylbenzamidine are catalysed by the same P450 isoenzyme. 4. A reaction mechanism is proposed in which the P450-dependent N-oxygenations and N-dealkylation of N-methylbenzamidine are derived from a common intermediate. It is obvious that if alpha-H atoms are present N-dealkylation is observed; however, in contrast with previous concepts, N-oxygenation is also possible.

Animals↗
PubMed1995

[Biotransformation processes in fertile hen's eggs. 1. Metabolic transformation of 7-ethoxycoumarin].

An alternative method for the investigation of biotransformations employing fertile chicken eggs has been developed. In these studies, solutions of 7-ethoxycoumarin were injected into the white of eggs from a standardized breed after brooding time of at least 8 days. After further incubation, the allantois was analyzed for metabolites of 7-ethoxycoumarin. The dealkylated metabolite of 7-ethoxycoumarin, namely 7-hydroxycoumarin, could be assayed in the allantois liquid by means of a new HPLC method. Furthermore, conjugates of 7-hydroxycoumarin, namely the glucuronid and the sulfate, can be detected indirectly in the allantois after conjugate cleavage. The inducability of the cytochrome P-450-dependent O-dealkylation has been studied with phenobarbital and 3-methylcholanthrene. An increase in the conversion of 7-ethoxycoumarin to 7-hydroxycoumarin was observed after addition of 3-methylcholanthrene. The detected metabolites demonstrate the usefulness of the developed model for studying biotransformation processes.

Animals↗
PubMed1994

CGRP is expressed in primary cultures of human hepatocytes and in normal liver.

We recently reported that human liver and primary cultures of hepatocytes express calcitonin. We therefore studied the expression of calcitonin gene related peptide (CGRP), the alternative splicing product of the calcitonin gene, in hepatocytes and liver. We used polymerase chain reaction amplification with specific primers to detect the presence of CGRP I and II messengers and a specific radioimmunoassay to measure the peptide. We report here that CGRP is synthesized by primary cultures of hepatocytes and in liver. As liver also possesses specific receptors for CGRP in non-parenchymal cells, a paracrine system could be involved in liver metabolism.

Alternative Splicing↗
PubMed1994

Enterocytic differentiation of the human Caco-2 cell line is correlated with down-regulation of fibronectin and laminin.

Human intestinal Caco-2 cells were used to examine the expression of fibronectin (FN) and laminin (LN) during enterocytic differentiation. Combination of immunoprecipitation, Western and Northern blotting revealed that Caco-2 cells expressed a classical FN and a variant form of LN: besides B1 and B2 chains, LN contained a 350-kDa heavy chain instead of the 400-kDa A chain. Throughout Caco-2 cell differentiation, FN and LN synthesis decreased at both mRNA and protein levels. These data indicate that enterocytic differentiation involves both transcriptional and/or post-transcriptional down-regulation of FN and LN gene expression.

Cell Differentiation↗
PubMed1994

Synthesis of 15N omega-hydroxy-L-arginine and ESR and 15N-NMR studies for the elucidation of the molecular mechanism of enzymic nitric oxide formation from L-arginine.

N omega-Hydroxy-L-arginine (2) was prepared by a multi-stage synthesis; the key step was the addition of hydroxylamine to the protected cyanamide 8. The presence of N-hydroxyguanidines was confirmed, above all, by 15N-NMR investigations. 15N omega-Hydroxy-L-arginine (2) was converted quantitatively to 15NO by NO synthases from macrophages. 15NO was identified by ESR-spectroscopy. These experiments confirm that 15N omega-hydroxy-L-arginine (2) is an intermediate in the biosynthesis of NO from arginine (1) and that the N-hydroxylated N-atom is present in the NO formed.

Amino Acid Oxidoreductases↗
PubMed1994

Influence of nidogen complexed or not with laminin on attachment, spreading, and albumin and laminin B2 mRNA levels of rat hepatocytes.

Nidogen/entactin is a Mr = 150,000 glycoprotein which is present within basement membranes in a noncovalent stable complex with laminin. We have studied the effects of nidogen/entactin complexed or not with laminin on attachment, spreading, and functions of adult rat hepatocytes in primary culture. Freshly isolated hepatocytes attached on either recombinant or EHS-derived nidogen, although to a lesser extent than on laminin/nidogen complex, laminin, and E8 and P1 fragments of laminin. Hepatocytes bound on a nidogen fragment bearing the N-terminal and rod-like domains but not on either the N-terminal globules or the rod-like domain which contains a RGD sequence. Attachment of hepatocytes on nidogen and laminin/nidogen complex was inhibited by anti-beta 1 integrin antibodies. Hepatocytes remained rounded on nidogen and laminin, whereas they rapidly spread on laminin/nidogen complex and collagen IV. Nidogen, laminin, and laminin/nidogen complex transiently maintained high steady-state albumin mRNA levels in cultured hepatocytes, but a decrease in albumin mRNA content was observed after 24 h, independently of the substrates. Actinomycin D and cycloheximide treatment indicated that the transient effect of these substrates on albumin expression was related to post-transcriptional mechanisms. Laminin B2 mRNAs were not detectable in freshly isolated hepatocytes but were expressed in 4 h hepatocyte cultures. After 24 h, a dramatic increase in the steady-state level of laminin B2 mRNA was found in hepatocytes cultured on nidogen and laminin/nidogen complex. This effect was slightly prevented in hepatocytes plated on laminin. These results show that interactions of hepatocytes with nidogen/entactin in vitro result only in a transient modulation of hepatocyte functions.

Animals↗
PubMed1994

Cytochrome P450-dependent N-hydroxylation of an aminoguanidine (amidinohydrazone) and microsomal retroreduction of the N-hydroxylated product.

1. The first example of a P450-dependent N-hydroxylation of an aminoguanidine (amidinohydrazone) is reported for 2-amino-5-chlorobenzophenone amidinohydrazone 1 (G 256) as substrate. 2. The N-hydroxylated metabolite 2 (2-amino-5-chlorobenzophenone N-hydroxyamidinohydrazone NOH-G256) and a further metabolite of 1, the phenol 3, were identified by tlc and ms analysis. 3. The microsomal reduction of an N-hydroxyaminoguanidine (N-hydroxy-amidino-hydrazone) was also demonstrated for the transformation of 2 to 1. 4. Both the N-hydroxylation of the aminoguanidine and the retroreduction of the N-hydroxyaminoguanidine were characterized by quantitative hplc analysis. 5. The conversion of the aminoguanidine 1 to N-hydroxyaminoguanidine 2 may be considered as an analogue of the physiological N-hydroxylation of arginine to N-hydroxyarginine by NO synthases.

Animals↗
PubMed1993

Cytochrome P450 dependent N-hydroxylation of a guanidine (debrisoquine), microsomal catalysed reduction and further oxidation of the N-hydroxy-guanidine metabolite to the urea derivative. Similarity with the oxidation of arginine to citrulline and nitric oxide.

The microsomal N-hydroxylation of the strongly basic guanidinium group (debrisoquine) to N-hydroxyguanidine (N-hydroxydebrisoquine) and the retroreduction of the N-hydroxyguanidine are demonstrated for the first time. The reduction of the N-hydroxyguanidine by liver homogenates and hepatocytes is catalysed by a microsomal NADH-dependent system that is strongly inhibited by hydroxylamine or N-methylhydroxylamine. In the presence of these alternate substrates for the reductase the microsomal catalysed N-hydroxylation of debrisoquine is readily characterized. The oxidation was inhibited by antibodies against NADPH cytochrome P450 reductase and the role of the P450 monooxygenase was further verified by studies with partially purified and purified P450 2C3 reconstituted systems. The transformation of N-hydroxydebrisoquine to the corresponding urea derivative was also detected in in vitro experiments with microsomal fractions and enriched P450 fractions as well as with flavin-containing monooxygenase (FMO). Experiments with catalase, superoxide dismutase and H2O2 have shown that the H2O2 or O2-, respectively, formed from the respective enzyme and the substrate, apparently participated in the reaction. Whereas the N-hydroxylation of the guanidine involves the usual monooxygenase activity of cytochrome P450 the resultant N-hydroxyguanidine decouples monooxygenases (cytochrome P450, FMO) and the H2O2 and, above all, O2- thus formed transform the N-hydroxyguanidine further to the corresponding urea derivative. The possibility for the N-hydroxylation of non-physiological guanidines to N-hydroxyguanidines and subsequent oxidative conversion to the respective urea is comparable to the physiological transformation of arginine to citrulline via N-hydroxyarginine with the liberation of nitric oxide (endothelial derived relaxing factor) and could, therefore, contribute to the efficacy of drugs containing guanidine and similar functional groups.

Animals↗
PubMed1993

Calcitonin gene expression in normal human liver.

Immunoreactive calcitonin (CT) is present in liver. This could represent hormone synthesized by liver cells, degraded or bound to specific receptors reported in this organ. We report here that the calcitonin gene is expressed in liver. We proved this by demonstrating, by PCR amplification using specific primers, the presence of calcitonin messenger in human liver and in primary cultures of human hepatocytes and detected by radioimmunoassay CT in hepatic tissues and cells. The synthesis of hormone by liver that also possesses specific receptors for CT favors the presence of an autocrine or paracrine system involving calcitonin in this organ.

Amino Acid Sequence↗
PubMed1993

Microsomal N-oxygenation of adenine to adenine 1-N-oxide.

During investigations on the N-oxygenation of adenine (1) the enzymatic formation of adenine 1-N-oxide 3 was demonstrated for the first time. The identity of this metabolite was confirmed by its chromatographic behaviour and UV-spectrum recorded after HPLC separation. Adenine 1-N-oxide (3) and similar oxygenated derivatives of adenine were synthesized as reference substances. The enzymatic formation of 3 exhibits the typical characteristics of a reaction catalysed by microsomal mono-oxygenases. In induction experiments, an increase in the rate of formation of 3 after pretreatment with phenobarbital was observed. A participation of those isoenzymes of the cytochrome P-450 enzyme system which can be induced by phenobarbital is assumed.

Adenine↗