Differential stereoselectivity of cytochromes P-450b and P-450c in the formation of naphthalene and anthracene 1,2-oxides. The role of epoxide hydrolase in determining the enantiomer composition of the 1,2-dihydrodiols formed

As is the case for cytochrome P-450c, arene 1,2-oxides have been identified as initial metabolites when naphthalene and anthracene are oxidized by cytochrome P-450b in a highly purified, reconstituted system. Overall rates of metabolism by cytochrome P-450b are greater than 3-fold and greater than 50-fold lower than the respective rates of metabolism by cytochrome P-450c. For both hydrocarbons, the (-)-(1S,2R)-oxide predominates (74%) with cytochrome P-450b as the terminal oxidant, based on trapping the labile arene oxides as N-acetyl-L-cysteine S-conjugates of known absolute configuration. This result is in marked contrast to data obtained with cytochrome P-450c where the (+)-(1R,2S)-oxides predominate (73-greater than 95%). In the absence of added epoxide hydrolase, the metabolically formed arene oxides rapidly isomerize to phenols. Addition of increasing amounts of epoxide hydrolase to the incubation medium results in the formation of trans-1,2-dihydrodiols at the expense of phenols from the common arene oxide intermediates. Evaluation of the kinetic parameters (Km and kcat) for the hydration of the (+)- and (-)-enantiomers of both arene oxides by epoxide hydrolase has indicated that the (+)-(1R,2S)-enantiomers exhibit lower values of Km (approximately 1 microM) whereas the values of kcat are similar for both enantiomers of a given arene oxide. These parameters have allowed construction of a mathematical model which predicts the enantiomer composition of the dihydrodiols formed from naphthalene in reconstituted systems containing specific epoxide hydrolase concentrations. The data reported argue against a selective functional coupling mechanism between cytochrome P-450c and epoxide hydrolase in the metabolism of naphthalene and anthracene to the 1,2-dihydrodiols.     

Mutagenicity of K-region epoxides of polycyclic aromatic compounds: Structure-activity relationship

The mutagenicity of several K-region arene oxides waas tested in histidine-dependent mutants of Salmonella typhimurium. Benzo(a)pyrene-4,5-oxide and pyrene-4,5-oxide as well as some substituted phenanthrene oxides were mutagenic in strains TA 1538 and TA 98 which detect frame-shift mutagens.Structure-activity relationships are discussed from the standpoint of chemical reactivity. The absence of direct correlation between electrophilic reactivity and mutagenicity may suggest that primilarily physical properties, such as relative position of the epoxide group and molecular shape of arene oxides, are important for the emergence of mutagenicity of arene oxides.     

Assay and partial purification of epoxide hydrase from rat liver microsomes

Solubilized cytochrome P-450 monooxygenase and epoxide hydrase activities from rat liver microsomes have been separated by column chromatography. The highly active epoxide hydrase fraction is still contaminated with cytochrome P-450, which has very low monooxygenase activity. The highly purified cytochrome P-450 fraction possesses high monooxygenase activity and is essentially devoid of epoxide hydrase activity. Purification factors for the epoxide hydrase through four purification steps are similar with [³H]styrene oxide, [³H]naphthalene oxide, [³H]cyclohexene oxide, and benzene oxide as substrates. Failure of benzene oxide to inhibit hydration of styrene or naphthalene oxide in the most purified preparations in indicative of the presence of at least two hydrases. These purified cytochrome monooxygenase and hydrase preparations represent valuable tools for the study of the intermediacy of arene oxides in drug metabolism. Thus, with naphthalene, only naphthol is formed with the monooxygenase, while both naphthol and the dihydrodiol are formed in the presence of monooxygenase and hydrase. A convenient radiochemical synthesis of [³H]naphthalene 1,2-oxide and assays for the measurement of the hydration of [³H]naphthalene oxide and benzene oxide, based on differential extractions and high-pressure liquid chromatography, respectively, are described.     

Determination of absolute configuration of conformationally flexible cis-dihydrodiol metabolites: effect of diene substitution pattern on the circular dichroism spectra and optical rotations

Absolute configurations of a number of cis-dihydrodiols (cis-1,2-dihydroxy-3,5-cyclohexadienes), synthetically useful products of TDO-catalyzed dihydroxylations of 1,2- and 1,3-disubstituted benzene derivatives, have been determined by a comparison of calculated and experimental CD spectra and optical rotations and by methods involving X-ray crystallography, 1H NMR spectra of diastereoisomeric derivatives, and by stereochemical correlations. The computations disclosed a significant effect of the substituents on conformational equilibria of cis-dihydrodiols and chiroptical properties of individual conformers. The assigned absolute configurations of cis-dihydrodiols have allowed the validity of a simple predictive model for TDO-catalyzed arene dihydroxylations to be extended.     

Biochemical evidence for the involvement of tyrosine in epoxide activation during the catalytic cycle of epoxide hydrolase

Epoxide hydrolases (EH) catalyze the hydrolysis of epoxides and arene oxides to their corresponding diols. The crystal structure of murine soluble EH suggests that Tyr(465) and Tyr(381) act as acid catalysts, activating the epoxide ring and facilitating the formation of a covalent intermediate between the epoxide and the enzyme. To explore the role of these two residues, mutant enzymes were produced and the mechanism of action was analyzed. Enzyme assays on a series of substrates confirm that both Tyr(465) and Tyr(381) are required for full catalytic activity. The kinetics of chalcone oxide hydrolysis show that mutation of Tyr(465) and Tyr(381) decreases the rate of binding and the formation of an intermediate, suggesting that both tyrosines polarize the epoxide moiety to facilitate ring opening. These two tyrosines are, however, not implicated in the hydrolysis of the covalent intermediate. Sequence comparisons showed that Tyr(465) is conserved in microsomal EHs. The substitution of analogous Tyr(374) with phenylalanine in the human microsomal EH dramatically decreases the rate of hydrolysis of cis-stilbene oxide. These results suggest that these tyrosines perform a significant mechanistic role in the substrate activation by EHs.     

Purification of human liver microsomal epoxide hydrase. Differences in the properties of the human and rat enzymes.

Human liver microsomal epoxide hydrase has been highly purified to a specific activity (570 to 620 nmol/min/mg of protein) comparable to that of the rat enzyme using styrene oxide as substrate. Like the purified rat liver microsomal epoxide hydrase, the human enzyme has a minimum molecular weight of 49,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and exhibits broad substrate specificity toward a variety of alkene and arene oxides. Despite these similarities, the human and rat enzymes are different proteins as judged by their immunochemical properties as well as their relative catalytic activities toward certain substrates.     

Stereoselective formation of benz[a]anthracene (+)-(5S,6R)-oxide and (+)-(8R,9S)-oxide by a highly purified and reconstituted system containing cytochrome P-450c

The principal oxidative metabolites formed from benz[a]anthracene (BA) by the rat liver microsomal monooxygenase system are the 5,6- and 8,9-arene oxides. In order to determine the enantiomeric composition and absolute configuration of these metabolically formed arene oxides, an HPLC procedure has been developed to separate the six isomeric glutathione conjugates obtained synthetically from the individual enantiomeric arene oxides. Both (+)- and (?)-BA 5,6-oxide gave the two possible positional isomers, but only one positional isomer was formed in each case from (+)- and (?)-BA 8,9-oxide. When [¹⁴C]-BA was incubated with a highly purified and reconstituted monooxygenase system containing cytochrome P-450c, and glutathione was allowed to react with the arene oxides formed, radio-active adducts were formed predominantly (>97%) from the (+)-(5S,6R) and (+)-(8R,9S) enantiomers. The present results are in accord with theoretical predictions of the steric requirements of the catalytic binding site of cytochrome P-450c.     

Spectroscopic investigation of the interaction of water-soluble azocalix[4]arenes with bovine serum albumin

In this work, three hydrosoluble azocalix[4]arene derivatives, 5-(o-methylphenylazo)-25,26,27-tris(carboxymethoxy)-28-hydroxycalix[4]arene (o-MAC-Calix), 5-(m-methylphenylazo)-25,26,27-tris(carboxymethoxy)-28-hydroxycalix[4]arene (m-MAC-Calix) and 5-(p-methylphenylazo)-25,26,27-tris(carboxymethoxy)-28-hydroxycalix[4]arene (p-MAC-Calix) were synthesized. Their structures were characterized by infrared spectrum (IR), nuclear magnetic resonance spectrum (¹H NMR and ¹³C NMR) and mass spectrum (MS). The interactions between these compounds and bovine serum albumin (BSA) were studied by fluorescence spectroscopy, UV–vis spectrophotometry and circular dichroic spectroscopy. According to experimental results, three azocalix[4]arene derivatives can efficiently bind to BSA molecules and the o-MAC-Calix displays more efficient interactions with BSA molecules than m-MAC-Calix and p-MAC-Calix. Molecular docking showed that the o-MAC-Calix was embedded in the hydrophobic cavity of helical structure of BSA molecular and the tryptophan (Trp) residue of BSA molecular had strong interaction with o-MAC-Calix. The fluorescence quenching of BSA caused by azocalix[4]arene derivatives is attributed to the static quenching process. In addition, the synchronous fluorescence spectroscopy indicates that these azocalix[4]arene derivatives are more accessible to Trp residues of BSA molecules than the tyrosine (Tyr) residues. The circular dichroic spectroscopy further verified the binding of azocalix[4]arene derivatives and BSA.     

Dinitroamino benzene derivatives: a class new potential high energy density compounds

Dinitroamino benzene derivatives are designed and studied in detail with quantum chemistry method. The molecular theory density, heats of formation, bond dissociation energies, impact sensitive and detonation performance are investigated at DFT-B3LYP/6-311G** level. The results of detonation performance indicated most of the compounds have better detonation velocity and pressure than RDX and HMX. The N-N bond can be regard as the trigger bond in explosive reaction, and the bond dissociation energies of trigger bond are almost not affected by the position and number of substituent group. The impact sensitive are calculated by two different theory methods. It is found that the compounds, which can become candidates of high energy materials, have smaller H₅₀ values than RDX and HMX. It is hoped that this work can provide some basis information for further theory and experiment studies of benzene derivatives.     

A rapid and reproducible assay for collagenase using [1-14C]acetylated collagen.

A rapid, continuous, and highly sensitive fluorescence assay is described for the measurement of epoxide hydrase activity. The method is based on the large differences between the fluorescence spectra of certain K-region arene oxides and their corresponding trans-dihydrodiols. Enzymatic hydration of K-region arene oxides of phenanthrene, pyrene, benzo[a]pyrene, and 7,12-dimethylbenzo[a]anthracene was studied. The assay was most sensitive with benzo[a]pyrene-4,5-oxide as substrate. With 10 μm benzo[a]pyrene-4,5-oxide, enzymatic rates of 30 pmol of dihydrodiol/min/mg of protein are three to five times those of the blank without enzyme. The fluorometric method described has been used to study site-directed inhibitors of epoxide hydrase and the stereoselective hydration of racemic arene oxides.     

Stereoselectivity of cytochrome P-450c in the formation of naphthalene and anthracene 1,2-oxides

Absolute configurations of the arene 1,2-oxides formed from napththalene and anthracene by cytochrome P-450c, the predominant isozyme of cytochrome P-450 found in the livers of rats treated with 3-methylcholanthrene, were determined via two different approaches. The first consisted of trapping the arene oxides with N-acetyl-L-cysteine to form S-conjugates, methylation of the conjugates with diazomethane, and separation of the resulting diastereomeric esters by reversed phase high performance liquid chromatography. Analysis by this procedure of the arene oxides formed from radioactive naphthalene and anthracene by a highly purified and reconstituted monooxygenase system containing cytochrome P-450c indicated that 73 and greater than or equal to 95%, respectively, of the metabolically formed arene oxides consisted of the (+)-(1R,2S)-enantiomer. In the second approach, each hydrocarbon was incubated with a reconstituted system containing both cytochrome P-450c and epoxide hydrolase. Under these conditions, the predominant metabolites are trans-1,2-dihydrodiols formed by epoxide hydrolase catalyzed trans-addition of water to the arene oxide intermediates. In both cases, the (-)-(1R,2R)-dihydrodiols predominated; 92% for naphthalene and 99% for anthracene. Enzyme-catalyzed addition of water to (+)- and (-)-anthracene 1,2-oxide and (+)-napthalene 1,2-oxide occurred exclusively (greater than 99%) at the allylic 2-position. The (-)-(1S,2R)-naphthalene 1,2-oxide, however, is converted to a 40:60 mixture of the (-)-(1R,2R)- and (+)-(1S,2S)-dihydrodiols by benzylic and allylic attack, respectively, resulting in increased enantiomeric purity of the dihydrodiol relative to the oxide. Thus, qualitatively and quantitatively both approaches indicate that the (+)-arene (1R,2S)-oxides predominate. The results are discussed in terms of the steric constraints of a proposed model for the catalytic binding site of cytochrome P-450c.     

Molecular orbital study of the hydroxylation of benzene and monofluorobenzene catalysed by iron-oxo porphyrin π cation radical complexes

 The reaction mechanism for the hydroxylation of benzene and monofluorobenzene, catalysed by a ferryl-oxo porphyrin cation radical complex (compound) is described by electronic structure calculations in local spin density approximation. The active site of the enzyme is modelled as a six-coordinated (Por⁺)Fe(IV)O a_2u complex with imidazole or H₃CS^– as the axial ligand. The substrates under study are benzene and fluorobenzene, with the site of attack in para, meta and ortho position with respect to F. Two reaction pathways are investigated, with direct oxygen attack leading to a tetrahedral intermediate and arene oxide formation as a primary reaction step. The calculations show that the arene oxide pathway is distinctly less probable, that hydroxylation by an H₃CS^––coordinated complex is energetically favoured compared with imidazole, and that the para position with respect to F is the preferred site for hydroxylation. A partial electron transfer from the substrate to the porphyrin during the reaction is obtained in all cases. The resulting charge distribution and spin density of the substrates reveal the transition state as a combination of a cation and a radical σ-adduct intermediate with slightly more radical character in the case of H₃CS^– as axial ligand. A detailed analysis of the orbital interactions along the reaction pathway yields basically different mechanisms for the modes of substrate–porphyrin electron transfer and rupture of the Fe–O bond. In the imidazole-coordinated complex an antibonding π*(Fe–O) orbital is populated, whereas in the H₃CS^––coordinated system a shift of electron density occurs from the Fe–O bond region into the Fe–S bond. Received: 1 July 1995 / Accepted: 18 December 1995     

Transformation of toluene and benzene by mixed methanogenic cultures

The aromatic hydrocarbons toluene and benzene were anaerobically transformed by mixed methanogenic cultures derived from ferulic acid-degrading sewage sludge enrichments. In most experiments, toluene or benzene was the only semicontinuously supplied carbon and energy source in the defined mineral medium. No exogenous electron acceptors other than CO2 were present. The cultures were fed 1.5 to 30 mM unlabeled or 14C-labeled aromatic substrates (ring-labeled toluene and benzene or methyl-labeled toluene). Gas production from unlabeled substrates and 14C activity distribution in products from the labeled substrates were monitored over a period of 60 days. At least 50% of the substrates were converted to CO2 and methane (greater than 60%). A high percentage of 14CO2 was recovered from the methyl group-labeled toluene, suggesting nearly complete conversion of the methyl group to CO2 and not to methane. However, a low percentage of 14CO2 was produced from ring-labeled toluene or from benzene, indicating incomplete conversion of the ring carbon to CO2. Anaerobic transformation pathways for unlabeled toluene and benzene were studied with the help of gas chromatography-mass spectrometry. The intermediates detected are consistent with both toluene and benzene degradation via initial oxidation by ring hydroxylation or methyl oxidation (toluene), which would result in the production of phenol, cresols, or aromatic alcohol. Additional reactions, such as demethylation and ring reduction, are also possible. Tentative transformation sequences based upon the intermediates detected are discussed.     

Transformation of toluene and benzene by mixed methanogenic cultures.

The aromatic hydrocarbons toluene and benzene were anaerobically transformed by mixed methanogenic cultures derived from ferulic acid-degrading sewage sludge enrichments. In most experiments, toluene or benzene was the only semicontinuously supplied carbon and energy source in the defined mineral medium. No exogenous electron acceptors other than CO2 were present. The cultures were fed 1.5 to 30 mM unlabeled or 14C-labeled aromatic substrates (ring-labeled toluene and benzene or methyl-labeled toluene). Gas production from unlabeled substrates and 14C activity distribution in products from the labeled substrates were monitored over a period of 60 days. At least 50% of the substrates were converted to CO2 and methane (greater than 60%). A high percentage of 14CO2 was recovered from the methyl group-labeled toluene, suggesting nearly complete conversion of the methyl group to CO2 and not to methane. However, a low percentage of 14CO2 was produced from ring-labeled toluene or from benzene, indicating incomplete conversion of the ring carbon to CO2. Anaerobic transformation pathways for unlabeled toluene and benzene were studied with the help of gas chromatography-mass spectrometry. The intermediates detected are consistent with both toluene and benzene degradation via initial oxidation by ring hydroxylation or methyl oxidation (toluene), which would result in the production of phenol, cresols, or aromatic alcohol. Additional reactions, such as demethylation and ring reduction, are also possible. Tentative transformation sequences based upon the intermediates detected are discussed.     

Stereoselective formation of benzo(c)phenanthrene (+)-(3S,4R) and (+)-(5S,6R)-oxides by cytochrome P450c in a highly purified and reconstituted system

The principal oxidative metabolites formed from benzo(c)phenanthrene (B(c)Ph) by the cytochromes P450 in liver microsomes from control and treated rats are the 3,4- and 5,6-arene oxides. A procedure is described which allows determination of the enantiomer composition and absolute configuration of these arene oxides based on HPLC separation of isomeric thiolate adducts formed with N-acetyl-L-cysteine in base. Incubation of [3H]-B(c)Ph with highly purified cytochrome P450c in a reconstituted monooxygenase system followed by trapping of the metabolically formed arene oxides as above indicated that the 3,4-oxide was predominantly the (+)-(3S,4R)-enantiomer (90%) and that the 5,6-oxide consisted mainly of the (+)-(5S,6R)-enantiomer (76%). The results are discussed in terms of their implications about the catalytic binding site of cytochrome P450c.     

Effect of benzamide derivatives on rat brain monoamine oxidase activity in vitro

The ability of moclobamide and other benzamide derivatives to inhibit the activity of monoamine oxidase in the rat brain was studied. Distinct effects of these compounds on the deamination of serotonin and norepinephrine (MAO-A substrates); 2-phenylethylamine (selective MAO-B substrate); tyramine and dopamine (MAO-A and MAO-B substrates) are shown. It was demonstrated that among all the compounds studied moclobamide appeared to be the most active and selective inhibitor of MAO-A: at a concentration of 100 microM it caused a 100% inhibition of serotonin and norepinephrine deamination, which might be explained by the presence of C1 atom in the para-position of benzene ring in moclobamide molecule. Other benzamide derivatives were less active in inhibiting MAO-A and had but a negligible effect on dopamine- and 2-phenylethylamine deamination.     

The role of stacking interactions in the binding of the NMDA receptor glycine site with antagonists and 3-hydroxykynurenine

Ab initio quantum chemical calculations of the benzene dimer, benzene dimer 5,7-chlorination of one aromatic ring, 3-hydroxykynurenine, and kynurenic acid molecules located above the Phe484 aromatic ring of a fragment of the receptor binding site were performed to study the role of stacking interaction in the binding of agonists and antagonists with the glycine binding site of the NR1 subunit of the NMDA receptor. The GAMESS 6.4 software in the 6–31G** basis set with complete optimization of the geometry and with account of electron correlation within the second-order Moller-Plesset perturbation theory was used for all calculations. It was shown that parallel shifted conformations of the benzene dimer were the most favorable in energy. Successive substitution of chlorine atoms for protons of one aromatic ring at positions 7 and 5 led to an increase in the stacking-interaction energy and mutual displacement of aromatic rings. In the case of kynurenic acid and its chlorinated derivatives, which are NMDA receptor antagonists, the increase in the stacking interaction energy further suppressed the ion channel, whereas 3-hydroxykynurenine was neither an agonist nor an antagonist of the glycine site because of steric constraints.     

A natural isolate of Pseudomonas maltophila which degrades aromatic sulfonic acids

Abstract A natural isolate, designated BSA56, which was originally selected for growth with benzene sulfuric acid as sole carbon and energy source, was identified as a strain of Pseudomonas maltophila . Strain BSA56 grew on a wide range of aromatic sulfonic acids and was shown to release sulfite from benzene sulfonic acid and 2-napthalene sulforic acid. Although it also grew on toluene sulfonic acid and pyridine sulfonic acid, no significant sulfite release was observed with these substrates. Release of sulfite from benzene sulfonic acid was greatly promoted by the presence of glycerol. The ability to release sulfite was induced by growth in the presence of benzene sulfonic acid and was repressed almost entirely by substrates allowing rapid growth such as acetate. Strain BSA56 grew better at 30°C than 37°C on most aromatic substrates, but the reverse was true for most aromatic sulfonates. Several mutants of BSA56 were isolated with defects in benzoate, salicylate, or gentisate metabolism. However, all these mutants retained the ability to degrade the aromatic sulfonates.