Cytochrome P450 2U1, a very peculiar member of the human P450s family.

Cytochrome P450 2U1 (CYP2U1) exhibits several distinctive characteristics among the 57 human CYPs, such as its presence in almost all living organisms with a highly conserved sequence, its particular gene organization with only five exons, its major location in thymus and brain, and its protein sequence involving an unusually long N-terminal region containing 8 proline residues and an insert of about 20 amino acids containing 5 arginine residues after the transmembrane helix. Few substrates, including fatty acids, N-arachidonoylserotonin (AS), and some drugs, have been reported so far. However, its biological roles remain largely unknown, even though CYP2U1 mutations have been involved in some pathological situations, such as complicated forms of hereditary spastic paraplegia. These data together with its ability to hydroxylate some fatty acids and AS suggest its possible role in lipid metabolism.

Brief historical overview and recent progress on cytochromes P450: adaptation of aerobic organisms to their chemical environment and new mechanisms of prodrug bioactivation.

The present brief overview of the history of the development of our knowledge on cytochromes P450 (P450s) illustrates the spectacular progress that have been made on P450 mechanisms and structures especially during these last 20 years. Recently published structures of mammalian P450-substrate complexes have shown the great diversity of size, shape, and binding modes that are offered by the conformationally flexible substrate binding sites of xenobiotic-metabolizing P450s. They have also shown that these binding sites can adapt themselves to the great structural diversity of xenobiotics, to facilitate their oxidation and elimination. Our present detailed knowledge of the mechanisms and chemistry of P450s allows us to understand, at the molecular level, the origin of the various consequences of P450-dependent metabolism of drugs in pharmacology and toxicology. This is here illustrated by recent data on the detailed mechanism of bioactivation of the anti-thrombotic prodrugs ticlopidine, clopidogrel, and prasugrel.

Antigenic targets in tienilic acid hepatitis. Both cytochrome P450 2C11 and 2C11-tienilic acid adducts are transported to the plasma membrane of rat hepatocytes and recognized by human sera.

Patients with tienilic acid hepatitis exhibit autoantibodies that recognize unalkylated cytochrome P450 2C9 in humans but recognize 2C11 in rats. Our aim was to determine whether the immune reaction is also directed against neoantigens. Rats were treated with tienilic acid and hepatocytes were isolated. Immunoprecipitation, immunoblotting, and flow cytometry experiments were performed with an anti-tienilic acid or an anti-cytochrome P450 2C11 antibody. Cytochrome P450 2C11 was the main microsomal or plasma membrane protein that was alkylated by tienilic acid. Inhibitors of vesicular transport decreased flow cytometric recognition of both unalkylated and tienilic acid-alkylated cytochrome P450 2C11 on the plasma membrane of cultured hepatocytes. Tienilic acid hepatitis sera that were preadsorbed on microsomes from untreated rats (to remove autoantibodies), poorly recognized untreated hepatocytes in flow cytometry experiments, but better recognized tienilic acid-treated hepatocytes. This recognition was decreased by adsorption with tienilic acid or by preexposure to the anti-tienilic acid or the anti-cytochrome P450 2C11 antibody. We conclude that cytochrome P450 2C11 is alkylated by tienilic acid and follows a vesicular route to the plasma membrane. Tienilic acid hepatitis sera contain antibodies against this tienilic acid adduct, in addition to the previously described anticytochrome P450 autoantibodies.

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.

Highly efficient control of iron-containing nitrile hydratases by stoichiometric amounts of nitric oxide and light.

The reaction of two iron-containing nitrile hydratases (NHase) with NO has been studied: NHase from Rhodococcus sp. R312, which is probably similar to the photosensitive N771 NHase, and the new NHase from Comamonas testosteroni NI1 whose aminoacid sequence is quite different from those of BR312 and N771 NHases. Both enzymes are equally inactivated after addition of stoichiometric amounts of NO added as an anaerobic solution or produced in situ under physiological conditions by a rat brain NO-synthase. Both enzymes are reactivated by photoirradiation, and two cycles of NO inactivation/photoactivation can be performed without significant loss of activity. Both iron-containing NHases have a high affinity for NO, similar to that of methemoglobin.

Studies of the reactivity of artificial peroxidase-like hemoproteins based on antibodies elicited against a specifically designed ortho-carboxy substituted tetraarylporphyrin.

The temperature and pH dependence as well as the selectivity of the peroxidase activity of a complex associating a monoclonal antibody 13G10 with its iron(III)-alpha,alpha,alpha,beta-mesotetrakis(ortho-carboxyphenyl) porphyrin (Fe(ToCPP)) hapten have been studied and compared to those of Fe(ToCPP) alone. It first appears that the peroxidase activity of the 13G10-Fe(ToCPP) complex is remarkably thermostable and remains about 5 times higher than that of Fe(ToCPP) alone until at least 80 degrees C. Secondly, this complex is able to use not only H2O2 as oxidant but also a wide range of hydroperoxides such as alkyl, aralkyl and fatty acid hydroperoxides and catalyze their reduction 2-6-fold faster than Fe(ToCPP) alone. It is also able to catalyze the oxidation by H202 of a variety of reducing cosubstrates such as 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD), 3,3',5,5'-tetramethylbenzidine (TMB) and 3,3'-dimethoxybenzidine 3-8-fold faster than Fe(ToCPP) alone, the bicyclic aromatic ABTS and TMB being the best reducing cosubstrates. Finally, a pH dependence study, between pH 4.6 and 7.5, of the oxidation of ABTS by H2O2 in the presence of either 13G10-Fe(ToCPP) or Fe(ToCPP) shows that Km(H2O2) values vary very similarly for both catalysts, whereas very different variations are found for the k(cat) values. With Fe(ToCPP) as catalyst the k(cat) value remains constant around 100 min(-1) whereas with the 13G10-Fe(ToCPP) complex, it increases sharply below pH 5 to reach 540 min -1 at pH 4.6. This could be due to the participation of a carboxylic acid side chain of the antibody protein, as a general acid-base catalyst, to the heterolytic cleavage of the O-O bond of H2O2 leading to the highly reactive iron(V)-oxo intermediate in the peroxidase mechanism. Accordingly, the modification of the carboxylic acid residues of antibody 13G10 by glycinamide leads to a 50% decrease of the peroxidase activity of the 13G10-Fe(ToCPP) complex.

Artificial peroxidase-like hemoproteins based on antibodies constructed from a specifically designed ortho-carboxy substituted tetraarylporphyrin hapten and exhibiting a high affinity for iron-porphyrins.

In order to get catalytic antibodies modelling peroxidases BALB/c mice have been immunized with iron(III)-alpha,alpha,alpha,beta-mesotetrakis-orthocarboxypheny l-porphyrin (Fe-(ToCPP))-KLH conjugates. Monoclonal antibodies have been produced by the hybridoma technology. Three antibodies, 2 IgG1 and 1 IgG2a, were found to bind both Fe(ToCPP) and the free base ToCPPH2 with similar binding constants. None of those antibodies was found to bind tetraphenylporphyrin. Those results suggest that the recognition of Fe(ToCPP) by the antibodies was mainly due to the binding of the carboxylate groups to some amino acid residues of the protein. True Kd values of 2.9 x 10(-9) M and 5.5 x 10(-9) M have been determined for the two IgG1-Fe(ToCPP) complexes. Those values are the best ones ever reported for iron-porphyrin-antibody complexes. UV-vis. studies have shown that the two IgG1-Fe(ToCPP) complexes were high-spin hexacoordinate iron(III) complexes, with no amino acid residue binding the iron, whereas the IgG2a-Fe(ToCPP) complex was a low-spin hexacoordinate iron(III) complex with two strong ligands binding the iron atom. Both IgG1-Fe(ToCPP) complexes were found to catalyze the oxidation of 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) 5-fold more efficiently than Fe(ToCPP) alone whereas the binding of IgG2a to this iron-porphyrin had no effect on its catalytic activity. kcat values of 100 min(-1) and 63 min(-1) and kcat/Km values of 105 M(-1) s(-1) and 119 M(-1) s(-1) have been found respectively for the two IgG1-Fe(ToCPP) complexes.

Bromocriptine is a strong inhibitor of brain nitric oxide synthase: possible consequences for the origin of its therapeutic effects.

The ergot alkaloid bromocriptine (BKT) was found to act as a strong inhibitor of purified neuronal nitric oxide synthase (NOS) (IC50 = 10 +/- 2 microM) whereas it was poorly active towards inducible macrophage NOS (IC50 > 100 microM). BKT affects the activation of NOS by calmodulin, as it not only inhibits L-arginine oxidation to NO and L-citrulline but also NADPH oxidation and calmodulin-dependent cytochrome c reduction catalyzed by neuronal NOS. These results suggest that BKT could exert some of its therapeutic effects by interfering with the NOS-dependent formation of nitric oxide and/or superoxide ion in various tissues.

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.

13C nuclear magnetic resonance studies of the binding of alkyl isocyanides to soybean leghemoglobin; comparison with animal myoglobins.

13C NMR of labelled alkyl isocyanide ligands has been used with a view to probe the protein environment around the heme site of Soybean leghemoglobin, and comparatively, those of sperm whale myoglobin and monomeric Glycera hemoglobin. The terminal carbon of the isocyanide, which is known to be highly sensitive to change in hybridization of the nitrogen, could be expected to reflect the movement of the alkyl group through steric interactions. Three alkyl isocyanides (alkyl = methyl, ethyl & n-butyl) have therefore been used and the 13C0 chemical shift values were measured for each ligand bound to the various proteins studied. In all cases, the 13C0 resonance of the bound ligand were shifted considerably down-field with respect to those of the free unbound species, but the pattern of these displacements revealed more pronounced steric hindrance in the case of some proteins compared to others. The modifications of the chemical shift values of binding delta delta = delta bound -- delta free) were least in the case of leghemoglobin; moreover, the delta delta values were insensitive to the length of the alkyl chain (methyl to n-butyl) when bound to leghemoglobin, in contrast to the other proteins examined. The results are interpreted as arising from a diminished steric hindrance to isocyanide binding with leghemoglobin, in conformity with the recently published X-ray structure which reports the existence of a large heme pocket on the distal side.

5-[4-(benzyloxy)phenyl]-1,3,4-oxadiazol-2(3H)-one derivatives and related analogues: new reversible, highly potent, and selective monamine oxidase type B inhibitors.

Thirty-three new 5-[4-(benzyloxy)phenyl]-1,3,4-oxadiazol-2(3H)-one derivatives including related analogues, designed as inhibitors of monoamine oxidase type B (MAO B), were synthesized and investigated both in vitro and ex vivo for their abilities to inhibit selectively rat brain MAO B over MAO A. Three inhibitors were found to act as reversible, highly potent, and selective MAO B inhibitors, namely the nitrile derivative 5-[4-(benzyloxy)phenyl]-3-(2-cyanoethyl)-1,3,4-oxadiazol-2(3H)-one (12a) and two closely related homologues, the corresponding oxadiazolethione 13a and the alcohol 14b. Their IC50 (MAO B) values are in the low nanomolar range of 1.4-4.6 nM and their selectivities, estimated by the ratio of IC50 values (A/B), are from 3200 to > 71,400. Compound 12a exhibited the highest activity against MAO B. Its IC50 was evaluated to be 1.4 nM with a quasitotal selectivity (> 71,400) toward this enzyme. In ex vivo studies, 12a showed a reversible and short duration of action. MAO B was markedly inhibited with the oral dose of 1 mg/kg without any alteration of MAO A, and the inhibition almost did not exceed 24 h. Its ED50 (1 h after oral administration) was evaluated to be 0.56 mg (1.7 mumol)/kg. Remarkably, MAO A was not affected at doses as high as 1500 mg/kg, po. In addition, no apparent toxicity or behavioral anomaly was observed during the treatment even at the maximum administrated dose. SAR studies emphasize the existence of three binding sites to the enzyme with a special importance of the terminal phenyl. Analysis of the inhibition kinetics indicated that 12a acts in a two-step mechanism as a competitive, slow, and tight-binding inhibitor of MAO B with a Ki value of 0.22 microM and an overall Ki* value at equilibrium of 0.7 nM.

Synthesis of sulfaphenazole derivatives and their use as inhibitors and tools for comparing the active sites of human liver cytochromes P450 of the 2C subfamily.

Twenty-three new derivatives of sulfaphenazole (SPA) were synthesized to further explore the topology of the active sites of human liver cytochromes P450 of the 2C subfamily and to find new selective inhibitors of these cytochromes. These compounds are derived from SPA by replacement of the NH(2) and H (of the SO(2)NH function) substituents of SPA with various R(1) and R(2) groups, respectively. Their inhibitory effects were studied on recombinant CYP 2C8, 2C9, 2C18, and 2C19 expressed in yeast. High affinities for CYP 2C9 (IC(50) < 1 microM) were only observed for SPA derivatives having the SO(2)NH function and a relatively small R(1) substituent (R(1) = NH(2), CH(3)). Any increase in the size of R(1) led to a moderate decrease of the affinity, and the N-alkylation of the SO(2)NH function of SPA to a greater decrease of this affinity. The same structural changes led to opposite effects on molecular recognition by CYP 2C8 and 2C18, which generally exhibited similar behaviors. Thus, contrary to CYP 2C9, CYP 2C8 and 2C18 generally prefer neutral compounds with relatively large R(1) and R(2) substituents. CYP 2C19 showed an even lower affinity for anionic compounds than CYP 2C8 and 2C18. However, as CYP 2C8 and 2C18, CYP 2C19 showed a much better affinity for neutral compounds derived from N-alkylation of SPA and for anionic compounds bearing a larger R(1) substituent. One of the new compounds (R(1) = methyl, R(2) = propyl) inhibited all human CYP 2Cs with IC(50) values between 10 and 20 microM, while another one (R(1) = allyl, R(2) = methyl) inhibited all CYP 2Cs except CYP 2C9, and a third one (R(1) = R(2) = methyl) inhibited all CYP 2Cs except CYP 2C8. Only 2 compounds of the 25 tested derivatives were highly selective toward one human CYP 2C; these are SPA and compound 1 (R(1) = CH(3), R(2) = H), which acted as selective CYP 2C9 inhibitors. However, some SPA derivatives selectively inhibited CYP 2C8 and 2C18. Since CYP 2C18 is hardly detectable in human liver, these derivatives could be interesting molecules to selectively inhibit CYP 2C8 in human liver microsomes. Thus, compound 11 (R(1) = NH(2), R(2) = (CH(2))(2)CH(CH(3))(2)) appears to be particularly interesting for that purpose as its IC(50) value for CYP 2C8 is low (3 microM) and 20-fold smaller than those found for CYP 2C9 and 2C19.

Structure-activity relationships of a new family of steroidal aromatase inhibitors. 1. Synthesis and evaluation of a series of analogs related to 19-[(methylthio)methyl]androstenedione (RU54115).

During the course of a study aimed at the search for new potent aromatase inhibitors, several new androstenedione analogs were synthesized and evaluated. This study led to the discovery of 19-[(methylthio)methyl]androsta-4,9(11)-diene-3,17-dione (7; RU54115) already described by our laboratory. The object of the present series of papers is to disclose the result of the structure-activity relationship studies that gave rise to this compound. This first part deals mainly with the substitution in the 19-position of the steroid nucleus. Several parameters were varied, the length of the chain and its rigidity and branching, as well as the nature of the heteroatom itself and its substitution. The interaction of these new compounds with human placental aromatase in competition with the substrate androstenedione was studied by difference visible spectroscopy. The in vivo aromatase-inhibiting activities were evaluated by measuring the estradiol lowering after oral administration of the compounds to PMSG-primed female rats.

Reduction of benzyl halides by liver microsomes. Formation of 478 NM-absorbing sigma-alkyl-ferric cytochrome P-450 complexes.

The benzyl halides benzyl bromide and 4-nitrobenzyl chloride are reduced anaerobically by NADPH and rat liver microsomes to yield toluene and 4-nitrotoluene, respectively. These reductions and cytochrome P-450-dependent since they are inhibited by CO and metyrapone, and are increased after pretreatment of rats by phenobarbital and 3-methylcholanthrene. During benzyl halide reduction, cytochrome P-450 complexes, which are very unstable to O2 and characterized by a Soret peak at 478 nm, are formed in steady-state concentrations. These concentrations are very dependent on pretreatment of rats and on the nature of the reducing agent (NADPH or dithionite) and the benzyl halide:4-methylbenzyl bromide and benzyl bromide lead to 478 nm absorbing complexes in the presence of NADPH whereas 4-nitrobenzyl chloride and benzyl chloride lead to such completes only in the presence of dithionite. Microsomal reductions of 4-nitrobenzyl chloride and benzyl bromide in D2O lead to partially deuterated 4-nitrotoluene and toluene. From these results, we propose a mechanism for anaerobic microsomal reduction of benzyl halides involving the intermediate formation of sigma-alkyl cytochrome P-450-Fe(III)-CH2Ar complexes which exhibit red-shifted Soret peaks around 478 nm. Toluenes, ArCH3, are formed either by protonation of the sigma-alkyl complexes or by hydrogen abstraction by the intermediate free radical ArCH2.

Particular ability of cytochromes P450 3A to form inhibitory P450-iron-metabolite complexes upon metabolic oxidation of aminodrugs.

The ability of 21 drugs containing an amine function to form inhibitory P450-iron-metabolite complexes absorbing around 455 nm was studied on liver microsomes from rats treated with various P450 inducers. These drugs belong to different chemical and therapeutic series and exhibit very different structures. In the case of eight compounds (diltiazem, lidocaine, imipramine, SKF 525A, fluoxetine, L-alpha-acetylmethadol, methadol and desmethyltamoxifen) whose oxidation by microsomes from rats treated with several inducers was studied, only dexamethasone (DEX)-treated rat microsomes and, to a lesser extent, phenobarbital (PB)-treated rat microsomes, were able to give significant amounts of 455 nm absorbing complexes. Ten of the 21 compounds studied gave such complexes with DEX-treated rat microsomes, while only three compounds gave complexes (in low amounts) with PB-treated rat microsomes only. For all compounds leading to complexes both with DEX- and PB-treated rat microsomes, much higher amounts of complexes were obtained with DEX-treated rat microsomes. DEX-treated rat microsomes also led to the most intense type I spectral interactions with most of the compounds studied, and very often exhibited the highest N-dealkylation activities towards the tertiary or secondary amine function of the drugs used. A few exceptions aside, there generally exists a qualitative relationship between the ability of P450 3As, induced by DEX, to bind and N-dealkylate amino compounds and their propensity to lead to 455 nm absorbing complexes. This was confirmed by in vivo experiments showing that rats treated with diltiazem, tamoxifen or imipramine accumulated large amounts of 455 nm absorbing complexes in their liver only after pretreatment with DEX and, to a lesser extent, with PB. This particular ability of P450 3As to oxidize amino drugs with formation of inhibitory P450-metabolite complexes could be of great importance for the appearance of drug interactions in man.

Dual effects of macrolide antibiotics on rat liver cytochrome P-450. Induction and formation of metabolite-complexes: a structure-activity relationship.

Previous studies have shown that the macrolide antibiotics, troleandomycin and erythromycin, are able to induce their own transformation into a metabolite forming an inactivated complex with rat liver cytochrome P-450. This paper reports the results of a study on the effects of several macrolide antibiotics including oleandomycin, erythromycin derivatives, josamycin, methymycin, tylosin, spiramycin and rifampicin, as well as antibiotics of other series, such as tetracycline and lincomycin, on rat liver cytochromes P-450 in vivo and in vitro. Only the antibiotics containing the desosamine and mycaminose amino sugars were able to give the dual effects already found with troleandomycin: induction of cytochrome p-450 and formation of an inhibitory cytochrome P-450--iron--nitrosoalkane metabolite complex in vivo or in vitro. From these studies, it appears that two structural factors are important for a macrolide antibiotic to lead to such effects: the presence of a non-hindered readily accessible N-dimethylamino group and the hydrophobic character of the molecule. These data are discussed in relation to the adverse effects observed during drug associations involving some of these macrolide antibiotics.

In vitro interaction of rat liver cytochromes P-450 with erythromycin, oleandomycin and erythralosamine derivatives. Importance of structural factors.

Several derivatives of the erythromycin, erythralosamine and oleandomycin series have been prepared. Their abilities to bind to rat liver microsomal cytochrome P-450 and to lead to the formation of stable 456 nm absorbing cytochrome P-450-metabolite complexes after their oxidative microsomal metabolism in vitro have been compared. The obtained data confirmed that cytochrome P-450 induced in rats either by macrolides or by 16 alpha-pregnenolone carbonitrile were the major isozymes involved in the binding of macrolides to liver microsomes and in metabolite-complex formation. They showed that (i) hydrophobicity was in general a beneficial factor for these two properties, (ii) the presence of a bulky substituent in position 3 of erythromycin dramatically decreased their affinity for these isozymes, and (iii) the simultaneous presence of bulky substituents in position 2' and 3 prevented iron-metabolite complex formation. These results led to the selection of two compounds, erythralosamine-2'-benzoate and erythralosamine-2',3-diacetate, which exhibited a particularly high affinity for macrolide inducible cytochrome P-450 and were very good precursors of cytochrome P-450-iron-metabolite complex formation.

Microsomal formation of nitric oxide and cyanamides from non-physiological N-hydroxyguanidines: N-hydroxydebrisoquine as a model substrate.

The microsomal oxidative transformation of a non-physiological N-hydroxyguanidine was demonstrated for the first time for N-hydroxydebrisoquine as a model substrate (Clement et al., Biochem Pharmacol 46: 2249-2267, 1993). The objective of the present work was to further compare this reaction with the analogous oxidation of arginine via N-hydroxyarginine to citrulline and nitric oxide. The oxidation of N-hydroxydebrisoquine by liver microsomes from rats pretreated with dexamethasone not only produced nitric oxide and the urea, but also the cyanamide derivative as the main metabolite. The low stability of the cyanamide derivative, which easily hydrolyzed to the urea derivative, was noted. The formation of all compounds required cosubstrate and the enzyme source. Experiments with catalase, superoxide dismutase, and H2O2 showed that the O2- formed from the enzyme and the substrate apparently participated in the reaction. While the N-hydroxylation of the guanidine involves the usual monooxygenase activity of cytochrome P-450 (Clement et al., Biochem Pharmacol 46: 2249-2267, 1993), the resultant N-hydroxyguanidine decoupled the monooxygenase. Nitric oxide was detected by the oxyhemoglobin assay. To examine the influence of enzymatically formed nitric oxide on the formation of the metabolites, the N-hydroxydebrisoquine was incubated with SIN-1 as nitric oxide donor under aerobic conditions. It was again possible to detect the cyanamide and urea derivatives, with the latter as main metabolite. It was concluded that the microsomal transformation of N-hydroxydebrisoquine produces a cyanamide and nitric oxide which reacts with N-hydroxydebrisoquine to form the urea derivative. The purely chemical reaction of the unsubstituted N-hydroxyguanidine with nitric oxide gave similar results (Fukuto et al., Biochem Pharmacol 43: 607-613, 1992). In conclusion, similarities (formation of a urea derivative) and differences (formation of a cyanamide derivative) between the physiological oxidation of N-hydroxy-L-arginine by nitric oxide synthases and non-physiological N-hydroxyguanidines by cytochrome P-450 were observed. Furthermore, non-physiological N-hydroxyguanidines can be regarded as nitric oxide donors.

Hydroxylation of the thiophene ring by hepatic monooxygenases. Evidence for 5-hydroxylation of 2-aroylthiophenes as a general metabolic pathway using a simple UV-visible assay.

The 5-hydroxylation of tienilic acid by rat liver microsomes was measured by a new, simple method involving the detection of 5-hydroxytienilic acid by UV-visible spectroscopy. This assay allowed continuous detection of this metabolite and could be easily used to determine the kinetic parameters of the reaction (Vmax and Km being respectively 1 +/- 0.2 nmol product formed/mg protein/min and 14 +/- 2 microM for liver microsomes from phenobarbital-treated rats). This activity was found to be dependent on NADPH and to be inhibited by CO, SKF 525A and metyrapone, indicating that it is dependent on cytochromes P-450. This UV-visible assay is based on intrinsic properties of 5-hydroxy 2-aroylthiophenes which exist as highly conjugated anions at physiological pH and exhibit large epsilon values around 390 nm. Its application to other 2-aroylthiophenes like suprofen, 2-parachlorobenzoylthiophene and a series of 2-aroylthiophenes with various substituents on the aroyl group showed that, in general, thiophene compounds bearing a 2-arylketo substituent appear to be hydroxylated at position 5 by rat liver microsomes. The kinetic parameters of the 5-hydroxylation of suprofen and 2-parachlorobenzoylthiophene by liver microsomes from phenobarbital-treated rats were determined and found to be similar to those for tienilic acid hydroxylation.

Oxidative activation of the thiophene ring by hepatic enzymes. Hydroxylation and formation of electrophilic metabolites during metabolism of tienilic acid and its isomer by rat liver microsomes.

Tienilic acid (TA) is metabolized by liver microsomes from phenobarbital-treated rats in the presence of NADPH with the major formation of 5-hydroxytienilic acid (5-OHTA) which is derived from the regioselective hydroxylation of the thiophene ring of TA. During this in vitro metabolism of TA, reactive electrophilic intermediates which bind irreversibly to microsomal proteins are formed. 5-Hydroxylation of TA and activation of TA to reactive metabolites which covalently bind to proteins both required intact microsomes, NADPH and O2 and are inhibited by metyrapone and SKF 525A, indicating that they are dependent on monooxygenases using cytochromes P-450. Microsomal oxidation of an isomer of tienilic acid (TAI) bearing the aroyl substituent on position 3 (instead of 2) of the thiophene ring also leads to reactive intermediates able to bind covalently to microsomal proteins. Covalent binding of TAI, as that of TA, depends on cytochrome P-450-dependent monooxygenases and is almost completely inhibited in the presence of sulfur containing nucleophiles such as glutathione, cysteine or cyteamine. These results show that 5-OHTA, which has been reported as the major metabolite of TA in vivo in humans, is formed by liver microsomes by a cytochrome P-450-dependent reaction. They also show that two thiophene derivatives, TA and TAI, bind to microsomal proteins after in vitro metabolic activation, TAI giving a much higher level of covalent binding than TA (about 5-fold higher) and a much higher covalent binding: stable metabolites ratio (4 instead of 0.5).