P450-catalyzed Oxidation Research Articles

Hydrogen atom abstraction of 3,5-disubstituted analogues of paracetamol by horseradish peroxidase and cytochrome P450

1. The formation of free radicals during enzyme catalysed oxidation of eight 3,5- disubstituted analogues of paracetamol (PAR) has been studied. A simple peroxidase system as well as cytochrome P450-containing systems were used. Radicals were detected by electron spin resonance (ESR) onincubationof PAR and 3,5-diCH-,3,5-diC H-,3,5- di t C H-, 3,5-diOCH-, 3,5-diSCH-, 3,5-diF-, 3,5-diCl- and 3,5-diBr-substituted analogues of PAR with horseradish peroxidase in the presence of hydrogen peroxide (H O). Initial analysis of the observed ESR spectra revealed all radical species to be phenoxy radicals, based on the absence of dominant nitrogen hyperfine splittings. No radicalswere detectedinrat livercytochromeP450-containingmicrosomalorreconstituted systems. 2. To rationalize the observed ESR spectra, hydrogen atom abstraction of PAR and four of the 3,5-disubstituted analogues (3,5-diCH-, 3,5-diOCH-, 3,5-diF- and 3,5-diClPAR) was calculated using ab initio calculations, and a singlet oxygen atom was used as the oxidizing species. The calculations indicated that for all compounds studied an initial hydrogen atomabstraction fromthe phenolic hydroxylgroup is favoured by approximately 125 kJ molover an initial hydrogen atom abstraction from the acetylamino nitrogen atom, and that after hydrogen abstraction from the phenolic hydroxyl group, the unpaired electron remains predominantly localised at the phenoxy oxygen atom (85%). 3. The experimental finding of phenoxy radicals in horseradish peroxidase H O incubations paralleled these theoretical findings. The failure to detect experimentally phenoxy radicals in cytochrome P450-catalysed oxidation of any of the eight 3,5- disubstituted PAR analogues is more likely due to the reducing effects that agents like NADPH and protein thiol groups have on phenoxy radicals rather than on the physical instability of the respective substrate radicals.

A Substituted Hypersensitive Radical Probe for Enzyme-Catalyzed Hydroxylations: Synthesis of Racemic and Enantiomerically Enriched Forms and Application in a Cytochrome P450-Catalyzed Oxidation

The syntheses of racemic and enantiomerically enriched trans-1-methyl-2-(4-(trifluoromethyl)phenyl)cyclopropane (3) and the possible oxidation products from enzyme-catalyzed hydroxylation of 3 at the methyl group are reported. The important intermediate in the production of 3 was the Weinreb amide of the 2-arylcyclopropanecarboxylic acid which could be prepared in diastereomerically pure form and which also served as an intermediate for production of the cyclic oxidation products of 3. Hydroxylation of 3 by the cytochrome P450 isozyme CYP2B1 gave cyclic and ring-opened products. The product ratios support an insertion mechanism for the enzyme-catalyzed hydroxylation reaction in which minor amounts of rearranged products are produced by radical fragmentation within the transition structure of the insertion and by a competing reaction involving a cationic species. Formation of cationic rearrangement products by a heterolytic fragmentation reaction of a first-formed protonated alcohol product is suggested on the basis of the apparent amounts of cationic products formed in the hydroxylation of 3. This pathway for cation production appears to require that the activated enzyme complex (equivalent to enzyme−substrate−H2O2) oxidizes substrate before water dissociates to give an iron−oxo species.

Potential metabolic bioactivation pathways involving cyclic tertiary amines and azaarenes.

A major theme explored in this review is the MAO-and cytochrome P450-catalyzed alpha-carbon oxidations of selected cyclic tertiary amines to give iminium metabolites that undergo further chemical modifications to form known or potentially toxic products. The most dramatic illustration of this type of bioactivation process is the conversion of the parkinsonian-inducing neurotoxin MPTP (23) by brain MAO-B to the iminium (dihydropyridinium) metabolite 24 which is oxidized further to the pyridinium species MPP+ (25). The selective destruction of nigrostriatal neurons by MPP+ is dependent on a unique sequence of events (transport into the nerve terminals by the dopamine transporter, localization in the inner mitochondrial membrane by electromotive forces, and inhibition of complex I of the mitochondrial electron transport chain) that, fortunately, are unlikely to be encountered with many substances. A second example of a well-documented metabolic bioactivation sequence involves the highly toxic pyrrolizidine alkaloids (102). These compounds undergo cytochrome P450-catalyzed alpha-carbon oxidation which converts the 3-pyrrolinyl moiety present in the parent alkaloids into a pyrrolyl-containing metabolite (105). The presence of labile functional groups results in the spontaneous conversion of 105 to reactive electrophilic products (106 and 108) that undergo Michael addition reactions with nucleophiles on biomacromolecules leading to a variety of toxic outcomes. Less clearly defined are the potential contributions to neurodegenerative processes that may be mediated by low-level, long term exposure to less potent toxins. Examples of potential proneurotoxins are the endogenously formed tetrahydroisoquinolines (such as 40-50) and tetrahydro-beta-carbolines (such as 54) that may be biotransformed to neurotoxic isoquinolinium (such as 51) and beta-carbolinium (such as 52) species in the brain. A similar argument can be made for 4-piperidinols (compounds that are at the same oxidation state as the tetrahydropyridines) which may be metabolized via iminium intermediates to amino enols that spontaneously convert to dihydropyridinium species and hence to pyridinium metabolites (67-->68-->69-->70-->71, Scheme 10). This type of reaction sequence has been well documented with the parkinsonian-inducing neuroleptic agent haloperidol (72) which is metabolized in humans, baboons, and rodents to the pyridinium species HPP+ (75), a potent inhibitor of mitochondrial respiration. Finally, an appreciation of the alpha-carbon oxidations of fully reduced azacycles such as (S)-nicotine (61) and phencyclidine (82) to chemically reactive metabolites that form covalent adducts with proteins, including the enzymes that are responsible for their formation, may prove of toxicological importance when attempting to account for the effects of chronic abuse of these potent drugs.1

1,2,3-Thiadiazole: a novel heterocyclic heme ligand for the design of cytochrome P450 inhibitors.

The 1,2,3-thiadiazole heterocycle has been explored as a heme ligand and mechanism-based inactivator for the design of cytochrome P450 inhibitors. One 4,5-fused bicyclic and three 4,5-disubstituted monocyclic 1,2,3-thiadiazoles have been examined for their spectral interactions, inhibition, mechanism-based inactivation, and oxidation products by the versatile microsomal P450s 2B4, 2E1, and 1A2. The compounds generally show heteroatom coordination to the heme iron; however, the binding mode is influenced by the architecture of the active site. For example, 4,5-diphenyl-1,2,3-thiadiazole shows type I and type II difference spectra with P450s 2B4 and 2E1, respectively, and no spectral perturbation with P450 1A2. Except for the fused bicyclic compound, the spectral dissociation constants are in the 2-50 microM range. The effectiveness as an inhibitor depends on the substituents at the 4- and 5- positions and on the P450 examined. Inhibition of the P450-catalyzed 1-phenylethanol oxidation to acetophenone by the thiadiazoles does not correlate with either the type of binding spectra or the spectral dissociation constants of the compounds. P450s 2E1 and 2B4 are inactivated by the 4,5-fused bicyclic 1,2,3-thiadiazole in a mechanism-based manner. Inactivation of the P450 correlates with loss in absorbance at 450 nm for the ferrous-CO complex. The monocyclic 1,2,3-thiadiazoles do not inactivate any of the P450s examined. The 1,2,3-thiadiazole ring is oxidized by the P450 system. Oxidation of the monocyclic compounds results in extrusion of the three heteroatoms and formation of the corresponding acetylenes, whereas oxidation of the fused bicyclic compound does not yield an acetylenic product.

Cytochrome P450-catalyzed oxidation of halobenzene derivatives.

ADVERTISEMENT RETURN TO ISSUEPerspectiveNEXTCytochrome P450-Catalyzed Oxidation of Halobenzene DerivativesIvonne M. C. M. Rietjens, Cathaline den Besten, Robert P. Hanzlik, and Peter J. van BladerenView Author Information Department of Biochemistry, Agricultural University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands, Department of Drug Safety, Solvay Duphar B.V., P.O. Box 900, 1381 CP Weesp, The Netherlands, Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas 66045, and Department of Toxicology, Agricultural University, Tuinlaan 5, 6703 HE Wageningen, and TNO Nutrition and Food Research Institution, Zeist, The Netherlands Cite this: Chem. Res. Toxicol. 1997, 10, 6, 629–635Publication Date (Web):June 16, 1997Publication History Received26 June 1996Published online16 June 1997Published inissue 1 June 1997https://doi.org/10.1021/tx9601061Copyright © 1997 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views783Altmetric-Citations52LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit Read OnlinePDF (231 KB) Get e-AlertsSUBJECTS:Aromatic compounds,Ethers,Hydrocarbons,Metabolism,Peptides and proteins Get e-Alerts

Direct Electrochemical Measurement of Nitric Oxide Production by Cytochrome P450-catalyzed Oxidation of N,N′-Substituted Hydroxyguanidines

The production of nitric oxide by oxidative cleavage of N- hydroxyguanidines catalyzed by rat liver microsomes was measured directly, and for the first time by differential-pulse amperometry at a Nafion–porphyrin coated micro-sensor. The measured concentration of the nitric oxide produced (0.18–0.25 µM) was lower than the total concentration of the stored nitrites. The difference in the obtained data is discussed.

The influence of 4-alkyl substituents on the formation and reactivity of 2-methoxy-quinone methides: evidence that extended π-conjugation dramatically stabilizes the quinone methide formed from eugenol

The effects of para-alkyl substituents on both the cytochrome P450-catalyzed oxidation of phenols to quinone methides (QMs; 4-methylene-2,5-cyclohexadien-1-ones), and on the rates of nucleophilic additions to the QMs were investigated. The derivatives of 4-alkyl-2-methoxyphenol studied were 4-methyl (creosol), 4-ethyl, 4-propyl, 4-isopropyl, and 4-allyl (eugenol). The relative reactivities of QMs derived from these phenols with water were 4-methyl > 4-ethyl = 4-propyl > 4-isopropyl > 4-allyl. These variations in rate were rationalized by differences in stabilization of positive charge density at the site of nucleophilic attack. In particular, saturation of the vinyl substituent on eugenol-QM increases the solvolysis rate 100-fold. This effect is presumably due to the loss of the contribution of an additional aromatic resonance structure to the overall resonance hybrid of the QM from 2-methoxy-4-propylphenol. Finally, the kinetic results show that there is a 472-fold difference in reactivity within this series of QMs. The QM glutathione conjugates were synthesized and characterized by 1H-NMR and electrospray mass spectrometry and a HPLC assay was developed to quantify QM formation in rat liver microsomes. The general trend is increasing alkyl substitution at the para position results in more QM; however, in contrast to the large range of reactivities of the QMs observed in the kinetic experiments, the amounts of P450-derived QM GSH adducts varied only by a factor of 3. In particular, similar amounts of the QMs from eugenol and 2-methoxy-4-propylphenol were produced which suggests that the lack of reported hepatotoxicity for the latter phenol in mice depleted of GSH, may be due to the extreme reactivity of 4-propyl-QM that would be rapidly detoxified by hydrolysis. These data suggest that there may be a threshold cytotoxicity level for QMs related to their reactivity which may affect the relative toxicities of 4-alkyl-2-methoxyphenols.

Differential induction of glutathione S-transferase in rat aorta versus liver

Polycyclic aromatic hydrocarbons, cigarette smoke components that induce atherosclerosis in animals, require metabolic biotransformation to electrophilic intermediates to exhibit atherogenic effects. The formation of reactive metabolites depends on both rates of cytochrome P450-catalyzed oxidation and rates of detoxification through conjugation with glutathione. Thus, changes in the activity of glutathione S-transferase in vascular tissue could affect the risk of polycyclic aromatic hydrocarbon-induced atherogenesis. We compared the effects of several exogenous chemicals on levels of glutathione S-transferase in aorta and liver. Male Wistar rats were treated with 3-methylcholanthrene, a polycyclic aromatic hydrocarbon, phenobarbital and butylated hydroxytoluene, an antioxidant known to have anti-atherogenic properties. In control animals, glutathione S-transferase activity was about 20-fold greater in liver than in aorta. Subunit expression was tissue specific. GST-Yp, for example, was the most abundant subunit in aorta but was undetectable in liver. In contrast, GST-Ya was barely detectable in aorta but was abundant in liver. Each of the xenobiotics caused induction of glutathione S-transferase but the extent of induction was greater in liver than in aorta. Phenobarbital, for example, caused 300% induction in liver but only 70% induction in aorta. By western blot analysis, differences in amounts of enzyme subunits corresponded to changes in enzyme activity. Thus, exogenous chemicals differentially regulate levels of glutathione S-transferase in the aorta and liver.

Kinetics of the Reduction of Cytochrome b5 with Mutations in Its Membrane-Binding Domain

In an attempt to understand which amino acids in the membrane anchor of cytochrome b5 might be determinants of its ability to support the cytochrome P450-catalyzed oxidation of selected substrates, the synthetic rat cytochrome b5 gene has been mutated by site-directed mutagenesis. The mutant proteins have been expressed in Saccharomyces cerevisiae, purified and assayed for their ability to support the cytochrome P450-catalyzed metabolism of the cytochrome b5 requiring substrate methoxyflurane (G. Vergères and L. Waskell, 1992, J. Biol. Chem. 267, 12583-12591). The rate of reduction of the cytochromes b5 by cytochrome P450 reductase has been examined by stopped-flow spectrophotometry to determine whether an altered rate of reduction of cytochrome b5 could explain the observed activity of cytochrome b5, in the purified reconstituted mixed-function oxidase system. A mutant in which the 22-amino-acid membrane anchor was replaced by a sequence of 22 leucines was unable to support methoxyflurane metabolism in the reconstituted system and was reduced by cytochrome P450 reductase at a rate (k = 4.5 × 10−3 s−1 slow enough to explain this finding. Comparison of the rate of reduction of this mutant cytochrome b5 in 0.025% Tergitol and 40 μM dilauroylphosphatidylcholine suggests that this slow rate of reduction may be explained partially by aggregation of the polyleucine protein. The Pro115Stop mutant protein, which has been truncated by 19 amino acids in its COOH terminus resulting in a protein with one-half of the putative membrane anchor, supports methoxyflurane oxidation at 12-20% of the rate of the wild type protein. In addition it is reduced by cytochrome P450 reductase at a rate which should be capable of supporting a normal rate of product formation. The fact that the Pro115Stop mutant,can be reduced at a rate capable of supporting a normal rate of methoxyflurane oxidation but in fact only supports methoxyflurane oxidation at 30% of the normal rate suggests that the mutant protein is deficient in its interactions with cytochrome P450. The mutant proteins, Pro115Ala and Ala116Pro, behaved essentially as did the wild type protein demonstrating that the presence (Pro115Ala) or absence (Ala116Pro) of an alpha helix in the middle of the putative membrane-binding domain of cytochrome b5 was not a determinant of the interaction of cytochrome b5 with cytochrome P450 reductase and cytochrome P450. The double mutant Ala131Lys;Glu132Lys in which the net negative charge at the C-terminus was altered to a net positive charge also functioned in a manner identical to that of the wild type protein, indicating that the net charge at the carboxyl terminus was not essential to the interaction of cytochrome b5 with its electron transfer partners.

Genetically determined adverse drug reactions involving metabolism.

Genetic factors represent an important source of interindividual variation in drug response. Relatively few adverse drug effects with a pharmacodynamic basis are known, and most of the well characterised inherited traits take the form of genetic polymorphisms of drug metabolism. Monogenic control of N-acetylation, S-methylation and cytochrome P450-catalysed oxidation of drugs can have important clinical consequences. Individuals who inherit an impaired ability to perform one or more of these reactions may be at an increased risk of concentration-related toxicity. There is a strong case for phenotyping before starting treatment with a small number of drugs that are polymorphically N-acetylated or S-methylated. However, the issue of clinical significance is perhaps most relevant for the debrisoquine oxidation polymorphism, which is mediated by cytochrome CYP2D6 and which determines the pharmacokinetics of many commonly used drugs. Phenotypic poor metabolisers of debrisoquine (8% of Caucasian populations) taking standard doses of some tricyclic antidepressants, neuroleptics or antiarrhythmic drugs may be particularly prone to adverse reactions. Similarly, clinically relevant drug interactions between these drugs and other substrates of cytochrome CYP2D6 may occur in the majority of the population who are extensive metabolisers. However, it is clear that in the majority of cases there is a need for controlled prospective studies to determine clinical significance. Accordingly, routine debrisoquine phenotyping or genotyping before beginning drug treatment is difficult to justify at present, although it may be helpful in individual cases. When prescribing drugs whose metabolism is polymorphic alone or in combination, careful titration of the dose in both phenotypic groups is prudent. In some cases it will be preferable to use alternative therapy to avoid the risk of adverse drug reactions.

Partitioning between N-dealkylation and N-oxygenation in the oxidation of N,N-dialkylarylamines catalyzed by cytochrome P450 2B1

Aminium radicals have been proposed as intermediates in amine N-dealkylations and N-oxygenations catalyzed by cytochrome P450 (P450) and some other enzymes. P450s can form some N-oxides and hydroxylamines but these are not favored whenever N-dealkylation is possible. However, if a paradigm involving 1-electron oxidation is valid, then some finite level of partitioning of aminium radicals between N-oxygenation and N-dealkylation might be expected in all cases. Methods were developed for the selective and sensitive measurement of N,N-dialkylaniline N-oxides using high performance liquid chromatography, radiochromatography, and TiCl3 reduction. These N-oxides were relatively stable in the presence of P450 2B1. In the presence of NADPH and NADPH-P450 reductase some reduction to N,N-dialkylamines occurred, along with N-dealkylation (to monoalkylanilines); there was also slow N-dealkylation in the absence of NADPH, which is interpreted in terms of homolytic scission of the N-O bond; N,N-dialkylanilines were not formed nor did the N-oxides support other oxygenation reactions. P450 2B1 (with its reductase and NADPH) formed N-oxides at low rates from several N,N-dialkylaniline derivatives, including N,N-dimethylaniline, N,N-diethylaniline, N-ethyl-N-methylaniline, 4-methyl-N,N-dimethylaniline, 4-cyano-N,N-dimethylaniline, N-phenylpyrrolidine, and N,N-dimethyl-2-aminofluorene. The ratio of N-dealkylation:N-oxygenation varied from 1020 to 6 in this series. These results are consistent with the view that aminium radicals are a branch point in N-oxygenation and N-dealkylation reactions catalyzed by metalloproteins, although some alternate explanations cannot be ruled out. While N-dealkylation is the dominant process in all of the P450-catalyzed amine oxidations, there should be a finite partition ratio between these reactions depending upon the particular enzyme and substrate. The N-oxygenation reaction is probably more complex than a direct radical recombination event and is postulated to involve one or more intermediates.

Cytochrome P450-related differences between rats and mice in the metabolism of benzene, toluene and trichloroethylene in liver microsomes

In evaluating the risks to humans of exposure to chemicals, the results of studies in rodents are sometimes used as a basis for extrapolation. It is therefore important to elucidate differences in metabolism among species. Differences in cytochrome P450-catalysed oxidation of benzene, toluene and trichloroethylene (TRI) between male Wistar rats and male B6C3F1 mice were investigated by immunoblot and immunoinhibition assays using monoclonal antibodies (MAbs) to cytochrome P450 (CYP1A1/2, CYP2B1/2, CYP2E1 and CYP2C11/6). Immunoblot analysis showed that anti-CYP2B1/2 did not detect any protein in either untreated rat or mouse liver microsomes, whereas with anti-CYP2E1 and/or anti-CYP1A1/2 a clear-cut band was seen more in liver microsomes from mice than from rats. Mouse liver microsomes had a greater monooxidation activity for benzene and TRI than rat liver microsomes; mice also had a higher rate of aromatic hydroxylation of toluene at low substrate concentration, but a low rate of side-chain oxidation when a high concentration of toluene was used. The metabolism of benzene was saturated in mice at around 0.23 mM, but the metabolism of the other two solvents was not saturated in either rats or mice at the low concentrations used. Anti-CYP2El inhibited the metabolism of benzene, toluene and TRI in microsomes from mice to a greater extent than in rats, while anti-CYP2C11/6 inhibited their metabolism in rats to a greater extent than in mice; anti-CYP1A1/2 inhibited the metabolism of TRI only in microsomes from mice. These results indicate that (i) male B6C3F1 mice have more CYP2E1 and 1A1/2 than male Wistar rats, whereas rats have more CYP2C11/6 than mice; (ii) rats and mice express CYP2B1/2 but they are not immunochemically detectable; (iii) CYP2E1 and 2C11/6 in both species are responsible for the metabolism of benzene, toluene and TRI, whereas CYP1A1/2 in mice catalyses the oxidation of TRI. The differences in the metabolism of benzene, toluene and TRI in rats and in mice may therefore depend, at least in part, on differences in the distribution of P450 isozymes between the two species.

The enzymic formation and chemical reactivity of quinone methides correlate with alkylphenol-induced toxicity in rat hepatocytes

The effects of o-alkyl substituents on both the cytochrome P450-catalyzed oxidation of phenols to p-quinone methides (QM's; 4-methylene-2,5-cyclohexadien-1-ones), and on the rates of nucleophilic additions to the 4-methylene carbon of QM's were investigated. The derivatives of 4-methylphenol studied were BHT (2,6-di-tert-butyl), BHTOH [6-tert-butyl-2-(hydroxy-tert-butyl)], BDMP (2-tert-butyl-6-methyl), BMP (2-tert-butyl), TMP (2,6-dimethyl), and DMP (2-methyl). QM formation was estimated to be in the range 0.17-0.70 nmol/(nmol of P450.min) in rat liver microsomes and 16-62 pmol/(10(6) cells.min) in isolated rat hepatocytes. QM's derived from BHT (BHT-QM), BHTOH (BHTOH-QM), BDMP (BDMP-QM), and TMP (TMP-QM) were synthesized and their rates of reaction with water and reduced glutathione (GSH) determined. BDMP-QM and TMP-QM were the most reactive, BHT-QM was consumed relatively slowly, and BHTOH-QM displayed intermediate reactivity. These variations in rate were rationalized by differences in hydrogen bonding with the carbonyl oxygen, which affects positive charge density at the site of nucleophilic attack. The loss of hepatocyte viability during incubations with BMP, BDMP, and BHTOH was preceded by GSH depletion. Pretreatment of hepatocytes with diethyl maleate exacerbated alkylphenol toxicity, and metyrapone protected the cells. These data, together with information on the formation and reactivity of QM's, strongly support the proposal that QM's mediate the toxicity of alkylated 4-methylphenols in rat hepatocytes.

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

Rat liver microsomes catalyze the oxidation of para-hexyloxybenzamidoxime 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 I 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.