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D Mansuy

Publications and source records attributed to D Mansuy.

At least 91 records · Page 5Linked to original sources
PubMed1993

Human-liver cytochromes P-450 expressed in yeast as tools for reactive-metabolite formation studies. Oxidative activation of tienilic acid by cytochromes P-450 2C9 and 2C10.

Human liver cytochromes P-450 (P450) 2C9 and 2C10 expressed in yeast reproduce all the metabolic features of the oxidation of tienilic acid (2-aryloxo-thiophene) and its isomer (3-aroylthiophene) by human liver microsomes. Microsomes of yeast expressing either P450 2C9 or P450 2C10 catalyze (a) the 5-hydroxylation of tienilic acid by NADPH and O2 (Km = 6 microM, Vmax = 2.5 turnover/min), (b) the activation of tienilic acid and its isomer into electrophilic metabolites which covalently bind to proteins, and (c) the formation of a mercaptoethanol adduct which results from the trapping of the tienilic acid isomer sulfoxide by this thiol. Microsomes of yeast expressing human liver P450 3A4, 1A1 and 1A2 are unable to catalyze these reactions. There is a striking similarity between the quantitative characteristics of the oxidation of tienilic acid (and its isomer) by yeast-expressed P450 2C9 (or 2C10) and by human liver microsomes: (a) analogous Km values (around 10 microM) for tienilic acid 5-hydroxylation, (b) a strong inhibition of tienilic acid oxidation by human sera containing anti-(liver kidney microsomes type 2) (anti-LKM2) antibodies, and (c) almost identical relative ratios of tienilic acid metabolic activation/5-hydroxylation and of tienilic acid activation/the activation of its isomer with both systems. Rates of oxidation of tienilic acid (and its isomer) by yeast microsomes are 6-8 fold higher than those found in human liver microsomes, which would be in agreement with the previously reported amount of P450 2C9 in human liver. These results not only suggest the important role of P450 2C9 in the oxidative metabolism of tienilic acid in human liver, but also indicate that the 5-hydroxylation reaction could be a useful marker for P450 2C9 activity and underline the interest of human liver P450s expressed in yeast as tools for studying the formation of reactive metabolites.

Biotransformation↗
PubMed1993

Escherichia coli and herpes-simplex-virus ribonucleotide reductase R2 subunit. Compared reactivities of the redox centres.

Protein R2, the small subunit of ribonucleotide reductase, contains a diferric centre and a tyrosyl radical absolutely required for enzyme activity. The reduction of the tyrosyl radical and the mobilization of the iron centre result in the inhibition of the enzyme and thus of DNA synthesis. The chemical reactivity of the iron-radical centre of Escherichia coli and herpes simplex virus has been studied by u.v.-visible and e.p.r. spectroscopies. The tyrosyl radical is efficiently scavenged by hydroxamic acids and phenols during reactions controlled by steric hindrance and hydrophobic interactions. The reaction with o-disubstituted phenols yields the corresponding diphenoquinones. The reactivity of the bacterial radical greatly contrasts with that of the viral radical, and the iron centre in herpes-simplex-virus R2 is much more labile than that in E. coli R2, as shown from the facile mobilization of iron by chelators such as catechol. These results suggest that the active sites of the two enzymes are significantly different and might be useful for designing new antiviral agents.

Binding Sites↗
PubMed1993

Particular ability of cytochrome P-450 CYP3A to reduce glyceryl trinitrate in rat liver microsomes: subsequent formation of nitric oxide.

Glyceryl trinitrate was denitrated in rat hepatic subcellular fractions, with formation of glyceryl dinitrates and glyceryl mononitrates. Among differently treated-rat liver microsomes, the highest microsomal activity was obtained under anaerobic conditions with microsomal preparations from dexamethasone-treated rats and NADPH. The reaction was inhibited by O2, CO, miconazole, dihydroergotamine and troleandomycin showing that it was catalyzed by cytochrome P-450 CYP3A isoforms. The formation of a transient cytochrome P-450 Fe(II)-NO complex during this reaction was shown by visible spectroscopy. The cytosolic activity was shown to be dependent on glutathione and glutathione transferase and was not inhibited by dioxygen. In the hepatic 9000 x g supernatant containing both NADPH and cytochrome P-450 and glutathione and glutathione transferase, the cytochrome P-450-dependent reaction accounts for 30-40% of the total denitration activity observed under anaerobic conditions, using 100 microM GTN.

Animals↗
PubMed1993

Structure-activity relationships for DNA photocleavage by cationic porphyrins.

The influence of molecular structure and DNA binding mode on the ability of cationic porphyrins to photosensitize DNA strand break formation has been studied for a series of meso-substituted pyridinium porphyrins using electrophoretic and DNA sequencing techniques. Porphyrins substituted with pyridyl groups in which the heterocyclic nitrogen is in the para or meta position vis-à-vis the substitution point are capable of intercalative binding and are considerably more efficient DNA photosensitizers than the corresponding non-intercalating ortho compounds. Within each group of porphyrins the photosensitizer efficiency increases with the number of positive charges. Using DNA sequencing experiments, we have demonstrated that photomodification occurs primarily at the guanine and thymine bases, and that alkali-labile sites produced by photo-oxidation are as important as direct cleavage events. The kinetics of strand degradation in aerated and degassed solution suggest that type II reactions (probably mediated by singlet oxygen) occur with significantly higher yield than type I reactions and are responsible for the formation of alkali-labile sites in aerated systems. These observations seem to confirm the hypothesis that those structural features which influence the strength and mode of binding also serve to establish favourable porphyrin-DNA interactions for photosensitization.

Base Sequence↗
PubMed1992

Cytochrome P450 catalyzes the oxidation of N omega-hydroxy-L-arginine by NADPH and O2 to nitric oxide and citrulline.

Rat liver microsomes catalyze the oxidative denitration of N omega-hydroxy-L-arginine (NOHA) by NADPH and O2 with formation of citrulline and nitrogen oxides like NO and NO2-. Besides NO2- and citrulline, whose simultaneous formation is linear for at least 20 min, the formation of NO could be detected under the form of its P450 and P420-Fe(II) complexes by UV-visible and EPR spectroscopy. Classical inhibitors of NO-synthases, like N omega-methyl-and N omega-nitro-arginine, fail to inhibit the microsomal oxidation of NOHA to citrulline and NO2-. On the contrary classical inhibitors of hepatic cytochromes P450 like CO, miconazole, dihydroergotamine and troleandomycin, strongly inhibit this monooxygenase reaction. These results show that the oxygenation of NOHA by NADPH and O2 with formation of citrulline and NO can be efficiently catalyzed by cytochromes P450 (with rates up to 1.5 turnovers per min for the cytochromes of the 3A subfamily).

Animals↗
PubMed1992

Evidence for thiophene-S-oxide as a primary reactive metabolite of thiophene in vivo: formation of a dihydrothiophene sulfoxide mercapturic acid.

Urine of rats treated with thiophene contains a very major metabolite which represents about 30% of the administered dose. A detailed analysis of its 1H and 13C NMR spectra and a study of its IR and mass spectra clearly showed that it was a 2,5-dihydrothiophene sulfoxide bearing a N-acetyl-cysteinyl group on position 2. Upon heating, it lost water with formation of N-acetyl-S-(2-thienyl)-L-cysteine. A likely mechanism for the formation of this metabolite should involve the S-oxidation of thiophene as a primary step and the addition of glutathione to the very reactive thiophene-S-oxide. These data provide a first evidence for the intermediate formation in vivo of thiophene-S-oxides as reactive metabolites.

Acetylcysteine↗
PubMed1992

Optimization of yeast-expressed human liver cytochrome P450 3A4 catalytic activities by coexpressing NADPH-cytochrome P450 reductase and cytochrome b5.

Human liver P450 NF25 (CYP3A4) had been previously expressed in Saccharomyces cerevisiae using the inducible GAL10-CYC1 promoter and the phosphoglycerate kinase gene terminator [Renaud, J. P., Cullin, C., Pompon, D., Beaune, P. and Mansuy, D. (1990) Eur. J. Biochem. 194, 889-896]. The use of an improved expression vector [Urban, P., Cullin, C. and Pompon, D. (1990) Biochimie 72, 463-472] increased the amounts of P450 NF25 produced/culture medium by a factor of five, yielding up to 10 nmol/l. The availability of recently developed host cells that simultaneously overexpress yeast NADPH-P450 reductase and/or express human liver cytochrome b5, obtained through stable integration of the corresponding coding sequences into the yeast genome, led to biotechnological systems with much higher activities of yeast-expressed P450 NF25 and with much better ability to form P450 NF25-iron-metabolite complexes. 9-fold, 8-fold, and 30-fold rate increases were found respectively for nifedipine 1,4-oxidation, lidocaine N-deethylation and testosterone 6 beta-hydroxylation between P450 NF25-containing yeast microsomes from the basic strain and from the strain that both overexpresses yeast NADPH-P450 reductase and expresses human cytochrome b5. Even higher turnovers (15-fold, 20-fold and 50-fold rate increases) were obtained using P450 NF25-containing microsomes from the yeast just overexpressing yeast NADPH-P450 reductase in the presence of externally added, purified rabbit liver cytochrome b5. This is explained by the fact that the latter strain contained the highest level of NADPH-P450 reductase activity. It is noteworthy that for the three tested substrates, the presence of human or rabbit cytochrome b5 always showed a stimulating effect on the catalytic activities and this effect was saturable. Indeed, addition of rabbit cytochrome b5 to microsomes from a strain expressing human cytochrome b5 did not further enhance the catalytic rates. The yeast expression system was also used to study the formation of a P450-NF25-iron-metabolite complex. A P450 Fe(II)-(RNO) complex was obtained upon oxidation of N-hydroxyamphetamine, catalyzed by P450-NF25-containing yeast microsomes. In microsomes from the basic strain expressing P450 NF25, 10% of the starting P450 NF25 was transformed into this metabolite complex, whereas more than 80% of the starting P450 NF25 led to complex formation in microsomes from the strain overexpressing yeast NADPH-P450 reductase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Formation of nitric oxide by cytochrome P450-catalyzed oxidation of aromatic amidoximes.

Rat liver microsomes catalyze the oxidation of para-hexyloxy-benzamidoxime 1 to the corresponding arylamide 2 and NO2-, by NADPH and O2. Involvement of cytochromes P450 as catalysts of this reaction was shown by the strong inhibitory effects of CO and miconazole and the spectacular increase of the activity upon treatment of rats with dexamethasone, a specific inducer of cytochromes P450 of the 3A subfamily. Formation of NO during oxidation of 1 was shown by detection of the formation of cytochrome P450- and cytochrome P420-Fe(II)-NO complexes by visible and EPR spectroscopy. The formation of these complexes should be responsible, at least in part, for the fast decrease of the rate of microsomal oxidation of 1 with time. These results suggest that exogenous compounds containing amidine or amidoxime functions could act as precursors of NO in vivo after in situ oxidation by cytochromes P450.

Animals↗
PubMed1992

Formation of nitrogen oxides and citrulline upon oxidation of N omega-hydroxy-L-arginine by hemeproteins.

HRP catalyzes the oxidation of N omega-hydroxy-L-arginine (NOHA) by H2O2 with formation of citrulline and NO2- with initial rates of about 0.7 and 0.2 nmol per nmol HRP per min. In the same manner, cytochromes P450 from rat liver microsomes catalyze the oxidation of NOHA to citrulline and NO2- by cumylhydroperoxide. Inhibitors of these hemeproteins (N3- and CN- for HRP and miconazole for P450) strongly inhibit both citrulline and NO2- formation. Rates of NOHA oxidation by these hemeproteins markedly decrease with time presumably because of their denaturation by nitrogen oxides and of the formation of hemeprotein-iron-NO complexes. These results suggest that NO (and other nitrogen oxides) could be formed from oxidation of NOHA by other enzymes than NO-synthases.

Animals↗
PubMed1992

In vitro metabolism of isaxonine phosphate: formation of two metabolites, 5-hydroxyisaxonine and 2-aminopyrimidine, and covalent binding to microsomal proteins.

Isaxonine phosphate or Nerfactor (2-isopropylaminopyrimidine) has been implicated in several cases of hepatitis which is reversible after withdrawal of the drug. In order to understand the cause of such hepatitis, the metabolic activation of isaxonine phosphate with different liver microsomes was investigated. The major metabolites were 5-hydroxyisopropylaminopyrimidine and 2-aminopyrimidine. Covalent binding to microsomal proteins was also detected. In vitro metabolic activation required intact microsomes, NADPH and O2 as cofactors and was cytochrome P-450 dependent. A sensitive fluorimetric assay of 5-hydroxyisaxonine was developed. The metabolism of isaxonine phosphate was compared in liver microsomes from rat, rabbit, dog, monkey and man and found to be qualitatively similar. Treatment of rats with phenobarbital increased the formation of 5-hydroxyisaxonine, while treatment with 3-methylcholanthrene increased the formation of 2-aminopyrimidine but decreased that of 5-hydroxyisaxonine. Inhibition and reconstitution experiments demonstrated that 5-hydroxylation of isaxonine was catalyzed by a cytochrome P-450. Metabolic oxidation of isaxonine phosphate using 5-[3H]isaxonine phosphate led to a total loss of tritium in 5-hydroxyisaxonine and partial loss of tritium in 2-aminopyrimidine and covalent binding to proteins.

Animals↗
PubMed1991

Mechanisms of inactivation of lipoxygenases by phenidone and BW755C.

Inhibition of soybean lipoxygenase (L-1) and potato 5-lipoxygenase (5-PLO) by the pyrazoline derivatives phenidone and BW755C only occurs after oxidation of these compounds by the peroxidase-like activity of the lipoxygenases. There is a clear relationship between this oxidation and the irreversible inactivation of L-1. The final product of phenidone oxidation by L-1, 4,5-didehydrophenidone, is not responsible of this inactivation, but the species derived from a one-electron oxidation of phenidone plays a key role in L-1 inactivation. In the absence of O2, inactivation of 1 mol of L-1 occurs after the oxidation of 34 mol of phenidone and the covalent binding of 0.8 mol of phenidone-derived metabolite(s) to L-1. In the presence of O2, inactivation of 1 mol of L-1 occurs already after oxidation of 11 mol of phenidone and only involves the covalent binding of 0.4 mol of phenidone-derived metabolite(s) to L-1. A mechanism is proposed for L-1 inactivation by phenidone, which involves the irreversible binding of a phenidone metabolite to the protein and the oxidation of an L-1 amino acid residue (in the presence of O2).

1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium ↗
PubMed1991

Metabolism of lipoxins A4 and B4 and of their all-trans isomers by human leukocytes and rat liver microsomes.

Incubation of lipoxin A4 (LXA4) either with human leukocytes or with rat liver microsomes in the presence of NADPH very selectively led to a more polar metabolite retaining the conjugated tetraenic structure of LXA4. Lipoxin B4 (LXB4) underwent a very similar metabolism into a more polar metabolite, whereas the all-trans isomers of LXA4 and LXB4 were selectively transformed by the same biological systems into metabolites derived from the reduction of one of the double bonds of the conjugated tetraene moiety of the starting compounds. Microsomal metabolism of LXA4 and LXB4 was NADPH-dependent and strongly inhibited by CO and miconazole indicating the involvement of cytochrome P-450 monooxygenase enzymes. Striking similarities between the metabolism of lipoxins and that of leukotriene B4 (LTB4) suggest that LXA4 and LXB4 are mainly hydroxylated, on omega or omega-I position, by human leukocytes and rat and human liver microsomes, whereas their all-trans isomers are mainly reduced into conjugated trienic compounds.

Animals↗
PubMed1991

Formation of prostaglandin synthase-iron-nitrosoalkane inhibitory complexes upon in situ oxidation of N-substituted hydroxylamines.

Various N-alkylhydroxylamines such as N-hydroxyamphetamine react with prostaglandin synthase (PGHS) from sheep seminal vesicles, with the formation of new complexes characterized by a Soret peak around 421 nm. These complexes are very stable toward O2 or dithionite but are destroyed upon oxidation by Fe(CN)6K3 with regeneration of starting PGHS-FeIII. Their spectral characteristics, chemical properties, and routes of formation (either by direct oxidation of RNHOH or by in situ reduction of RNO2 in the presence of dithionite) are very similar to those previously reported for nitrosoalkane complexes of hemoglobin-, myoglobin-, and cytochrome P-450-FeII. Their FeII-N(O)R structure was completely confirmed in the case of N-hydroxyamphetamine, both by extraction of the heme complex by butanone and by identification to authentic protoporphyrin IX-FeII-N(O)-amphetamine, and by insertion of this authentic complex into apoPGHS. Phenylhydroxylamine also reacts with PGHS-FeIII to give a PGHS-FeII-N(O)Ph complex which is not stable in the presence of dithionite because of its weaker PGHS-FeII-N(O)R bond when compared to PGHS-FeII-nitrosoalkane complexes. The ability of various N-alkylhydroxylamines to form PGHS-FeII-N(O)R complexes greatly depends upon their hydrophobicity. Actually, CH3NHOH and C2H5NHOH are totally inactive whereas about 10 molar excess of N-hydroxyamphetamine and C6H5NHOH already lead to 50% complex formation. This is in favor of an hydrophobic environment of the heme in PGHS. Finally, PGHS engaged in such FeII-nitrosoalkane complexes completely loses its dioxygenase activity, suggesting that N-substituted hydroxylamines or compounds that can be metabolized in vivo to give such hydroxylamines could act as strong PGHS inhibitors.

Animals↗
PubMed1991

Hydroxylation and formation of electrophilic metabolites of tienilic acid and its isomer by human liver microsomes. Catalysis by a cytochrome P450 IIC different from that responsible for mephenytoin hydroxylation.

Tienilic acid (TA) is metabolized by human liver microsomes in the presence of NADPH with the major formation of 5-hydroxytienilic acid (5-OHTA) which is derived from the hydroxylation of the thiophene ring of TA. Besides this hydroxylation, TA is oxidized into reactive metabolites which covalently bind to microsomal proteins. Oxidation of an isomer of tienilic acid (TAI), bearing the aroyl substituent on position 3 (instead of 2) of the thiophene ring, by human liver microsomes, gives a much higher level of covalent binding to proteins. Both covalent binding of TA and TAI metabolites are almost completely suppressed in the presence of glutathione. These three activities of human liver microsomes (TA 5-hydroxylation, covalent binding of TA and TAI metabolites) seem dependent on the same cytochrome P450 of the IIC subfamily, since (i) antibodies against human liver cytochromes P450 IIC strongly inhibit these three activities, (ii) there is a clear correlation between these activities in various human liver microsomes, and (iii) TA acts as a competitive inhibitor for TAI activation into electrophilic metabolites (Ki approximately equal to 25 microM) and TAI inhibits TA 5-hydroxylation. However cross inhibition experiments indicate that tienilic acid hydroxylation and mephenytoin hydroxylation, a typical reaction of some human liver P450 IIC isoenzymes, are not catalysed by the same member of the P450 IIC subfamily.

Biotransformation↗
PubMed1991

Vitamin E derivatives as new potent inhibitors of microsomal lipid peroxidation.

Several synthetic Vitamin E derivatives are strong inhibitors of lipid peroxidation induced in rat liver microsomes either chemically by ferrous ions and ascorbate or enzymatically by NADPH and carbon tetrachloride. The relative activities of these inhibitors are consistent with their intrinsic antioxidant properties, as peroxyl radicals scavengers. Among them, a 3,4-dihydro-6-hydroxy-2H-1-naphtopyran with IC50 around 0.08 microM is one of the most potent yet known inhibitor of lipid peroxidation.

Animals↗
PubMed1991

Peroxidase-like activity of lipoxygenases: different substrate specificity of potato 5-lipoxygenase and soybean 15-lipoxygenase and particular affinity of vitamin E derivatives for the 5-lipoxygenase.

Potato 5-lipoxygenase (5-PLO) catalyzes the reduction of 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid (13-HPOD) in the presence of vitamin E. I mol of vitamin E is required to consume 2 mol of 13-HPOD. The mechanism of the 5-PLO-catalyzed oxidation of vitamin E by 13-HPOD is similar to that previously established for the soybean 15-lipoxygenase (L-1)-catalyzed oxidation of phenidone by 13-HPOD, and seems to involve a one-electron reduction of the O-O bond of 13-HPOD. 5-PLO and L-1 exhibit very different substrate specificities and pH profiles for their peroxidase-like activity. Actually, among the 20 compounds containing various reducible functions and the 10 derivatives of vitamin E which have been studied, only four products containing hydrophobic long chains, ascorbic acid 6-palmitate, the trolox esters of octanol and undecanol, and vitamin E exhibit high peroxidase-like activities for 5-PLO. On the contrary, much more compounds, even not very hydrophobic, are good substrates for the peroxidase-like activity of L-1.

Arachidonate 15-Lipoxygenase↗
PubMed1991

Detection of human hepatitis anti-liver kidney microsomes (LKM2) autoantibodies on rat liver sections is predominantly due to reactivity with rat liver P-450 IIC11.

Anti-liver kidney microsomes (anti-LKM2) autoantibodies, appearing in patients treated with tienilic acid and suffering from hepatitis, react with proteins in rat liver sections. The nature of the rat proteins responsible for this recognition and detection of anti-LKM2 has been investigated. Immunoblot testing of the anti-LKM2 with liver microsomes from diversely treated rats and with purified rat liver cytochromes P450 (IA1, IA2, IIB1, IIB2, IIC6, IIC11 and IVA1) showed that these antibodies cross-reacted with cytochrome P450IIC11 and also with phenobarbital-induced cytochromes P450IIB1 and IIB2. Moreover, metabolic activation of tienilic acid and of a tienilic acid isomer by untreated rat liver microsomes was partially inhibited by anti-LKM2. On the other hand, monospecific polyclonal anti-rat P450IIC11 antibodies cross-reacted with human microsomal cytochromes P450 and recognized the same cytochromes P450 as anti-LKM2. This antibody also gave an immunofluorescence pattern on rat and mouse liver and kidney sections very similar to anti-LKM2. The data presented here show that anti-LKM2 recognize epitopes shared by rat P450 IIC11, and a human P450 of the family IIC. All the results indicate rat P450 IIC11, the major isoenzyme present in normal adult male rat liver, as the main antigen recognized by human anti-LKM2 autoantibodies; this is the basis of the immunofluorescence test for detection of these antibodies.

Animals↗