Browse
Submit Data
Databases
API
Help

Dataset Information

10 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Roles of cytochrome P450 2A6 in the oxidation of flavone, 4'-hydroxyflavone, and 4'-, 3'-, and 2'-methoxyflavones by human liver microsomes.

ABSTRACT: Nine forms of recombinant cytochrome P450 (P450 or CYP) enzymes were used to study roles of individual P450 enzymes in the oxidation of flavone and some other flavonoids, 4'-hydroxyflavone and 4'-, 3'-, and 2'-methoxyflavones, by human liver microsomes using LC-MS/MS analysis.As has been reported previously , 4'-, 3'-, and 2'-methoxyflavones were preferentially O-demethylated by human liver P450 enzymes to form 4'-, 3'-, and 2'-hydroxylated flavones and also 3',4'-dihydroxyflavone from the former two substrates.In comparisons of product formation by oxidation of these methoxylated flavones, CYP2A6 was found to be a major enzyme catalysing flavone 4'- and 3'-hydroxylations by human liver microsomes but did not play significant roles in 2'-hydroxylation of flavone, O-demethylations of three methoxylated flavones, and the oxidation of 4'-hydroxyflavone to 3',4'-dihydroxyflavone.The effects of anti-CYP2A6 IgG and chemical P450 inhibitors suggested that different P450 enzymes, as well as CYP2A6, catalysed oxidation of these flavonoids at different positions by liver microsomes.These studies suggest that CYP2A6 catalyses flavone 4'- and 3'-hydroxylations in human liver microsomes and that other P450 enzymes have different roles in oxidizing these flavonoids.

SUBMITTER: Nagayoshi H

PROVIDER: S-EPMC8719450 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Site-specific oxidation of flavanone and flavone by cytochrome P450 2A6 in human liver microsomes.

Project description:The roles of human cytochrome P450 (P450 or CYP) 2A6 in the oxidation of flavanone [(2R)- and (2S)-enantiomers] and flavone were studied in human liver microsomes and recombinant human P450 enzymes. CYP2A6 was highly active in oxidizing flavanone to form flavone, 2'-hydroxy-, 4'-, and 6-hydroxyflavanones and in oxidizing flavone to form mono- and di-hydroxylated products, such as mono-hydroxy flavones M6, M7, and M11 and di-hydroxy flavones M3, M4, and M5. Liver microsomes prepared from human sample HH2, defective in coumarin 7-hydroxylation activity, were very inefficient in forming 2'-hydroxyflavanone from flavanone and a mono-hydroxylated product, M6, from flavone. Coumarin and anti-CYP2A6 antibodies strongly inhibited the formation of these metabolites in microsomes prepared from liver samples HH47 and 54, which were active in coumarin oxidation activities. Molecular docking analysis showed that the C2'-position of (2R)-flavanone (3.8 Å) was closer to the iron center of CYP2A6 than the C6-position (10 Å), while distances from C2' and C6 of (2S)-flavanone to the CYP2A6 were 6.91 Å and 5.42 Å, respectively. These results suggest that CYP2A6 catalyzes site-specific oxidation of (racemic) flavanone and also flavone in human liver microsomes. CYP1A2 and CYP2B6 were also found to play significant roles in some of the oxidations of these flavonoids by human liver microsomes.

| S-EPMC6438780 | biostudies-literature

Cytochrome P450 2A6 and other human P450 enzymes in the oxidation of flavone and flavanone.

Project description:1. We previously reported that flavone and flavanone interact spectrally with cytochrome P450 (P450 or CYP) 2A6 and 2A13 and other human P450s and inhibit catalytic activities of these P450 enzymes. In this study, we studied abilities of CYP1A1, 1A2, 1B1, 2A6, 2A13, 2C9 and 3A4 to oxidize flavone and flavanone. 2. Human P450s oxidized flavone to 6- and 5-hydroxylated flavones, seven uncharacterized mono-hydroxylated flavones, and five di-hydroxylated flavones. CYP2A6 was most active in forming 6-hydroxy- and 5-hydroxyflavones and several mono- and di-hydroxylated products. 3. CYP2A6 was also very active in catalyzing flavanone to form 2'- and 6-hydroxyflavanones, the major products, at turnover rates of 4.8 min-1 and 1.3 min-1, respectively. Other flavanone metabolites were 4'-, 3'- and 7-hydroxyflavanone, three uncharacterized mono-hydroxylated flavanones and five mono-hydroxylated flavones, including 6-hydroxyflavone. CYP2A6 catalyzed flavanone to produce flavone at a turnover rate of 0.72 min-1 that was ∼3-fold higher than that catalyzed by CYP2A13 (0.29 min-1). 4. These results indicate that CYP2A6 and other human P450s have important roles in metabolizing flavone and flavanone, two unsubstituted flavonoids, present in dietary foods. Chemical mechanisms of P450-catalyzed desaturation of flavanone to form flavone are discussed.

| S-EPMC6430112 | biostudies-literature

Mechanism-based inhibition of cytochrome P450 (CYP)2A6 by chalepensin in recombinant systems, in human liver microsomes and in mice in vivo.

Project description:Background and purposeChalepensin is a pharmacologically active furanocoumarin compound found in rue, a medicinal herb. Here we have investigated the inhibitory effects of chalepensin on cytochrome P450 (CYP) 2A6 in vitro and in vivo.Experimental approachMechanism-based inhibition was studied in vitro using human liver microsomes and bacterial membranes expressing genetic variants of human CYP2A6. Effects in vivo were studied in C57BL/6J mice. CYP2A6 activity was assayed as coumarin 7-hydroxylation (CH) using HPLC and fluorescence measurements. Metabolism of chalepensin was assessed with liquid chromatography/mass spectrometry (LC/MS).Key resultsCYP2A6.1, without pre-incubation with NADPH, was competitively inhibited by chalepensin. After pre-incubation with NADPH, inhibition by chalepensin was increased (IC(50) value decreased by 98%). This time-dependent inactivation (k(inact) 0.044?min(-1) ; K(I) 2.64?µM) caused the loss of spectrally detectable P450 content and was diminished by known inhibitors of CYP2A6, pilocarpine or tranylcypromine, and by glutathione conjugation. LC/MS analysis of chalepensin metabolites suggested an unstable epoxide intermediate was formed, identified as the corresponding dihydrodiol, which was then conjugated with glutathione. Compared with the wild-type CYP2A6.1, the isoforms CYP2A6.7 and CYP2A6.10 were less inhibited. In mouse liver microsomes, pre-incubation enhanced inhibition of CH activity. Oral administration of chalepensin to mice reduced hepatic CH activity ex vivo.Conclusions and implicationsChalepensin was a substrate and a mechanism-based inhibitor of human CYP2A6. Formation of an epoxide could be a key step in this inactivation. 'Poor metabolizers' carrying CYP2A6*7 or *10 may be less susceptible to inhibition by chalepensin. Given in vivo, chalepensin decreased CYP2A activity in mice.

| S-EPMC3144538 | biostudies-literature

Nanobioelectrocatalysis Using Human Liver Microsomes and Cytochrome P450 Bactosomes: Pyrenyl-Nanocarbon Electrodes.

Project description:Human liver microsomes containing various drug-metabolizing cytochrome P450 (P450) enzymes, along with their NADPH-reductase bound to phospholipid membranes, were absorbed onto 1-pyrene butylamine pi-pi stacked with amine-functionalized multiwalled carbon nanotube-modified graphite electrodes. The interfaced microsomal biofilm demonstrated direct electrochemical communication with the underlying electrode surface and enhanced oxygen reduction electrocatalytic activity typical of heme enzymes such as P450s over the unmodified electrodes and nonenzymatic currents. Similar enhancements in currents were observed when the bioelectrodes were constructed with recombinant P450 2C9 (single isoform) expressed bactosomes. The designed liver microsomal and 2C9 bactosomal bioelectrodes successfully facilitated the electrocatalytic conversion of diclofenac, a drug candidate, into 4'-hydroxydiclofenac. The enzymatic electrocatalytic metabolite yield was several-fold greater on the modified electrodes than on the unmodified bulk graphite electrodes adsorbed with a microsomal or bactosomal film. The nonenzymatic metabolite production was less than the enzymatically catalyzed metabolite yield in the designed microsomal and bactosomal biofilm electrodes. To test the throughput potential of the designed biofilms, eight-electrode array configurations were tested with the microsomal and bactosomal biofilms toward electrochemical 4'-hydroxydiclofenac metabolite production from diclofenac. The stability of the designed microsomal bioelectrode was assessed using nonfaradaic impedance spectroscopy over 40 h, which indicated good stability.

| S-EPMC11022171 | biostudies-literature

Metabolic capabilities of cytochrome P450 enzymes in Chinese liver microsomes compared with those in Caucasian liver microsomes.

Project description:AimThe most common causes of variability in drug response include differences in drug metabolism, especially when the hepatic cytochrome P450 (CYP) enzymes are involved. The current study was conducted to assess the differences in CYP activities in human liver microsomes (HLM) of Chinese or Caucasian origin.MethodsThe metabolic capabilities of CYP enzymes in 30 Chinese liver microsomal samples were compared with those of 30 Caucasian samples utilizing enzyme kinetics. Phenacetin O-deethylation, coumarin 7-hydroxylation, bupropion hydroxylation, amodiaquine N-desethylation, diclofenac 4'-hydroxylation (S)-mephenytoin 4'-hydroxylation, dextromethorphan O-demethylation, chlorzoxazone 6-hydroxylation and midazolam 1'-hydroxylation/testosterone 6?-hydroxylation were used as probes for activities of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A, respectively. Mann-Whitney U test was used to assess the differences.ResultsThe samples of the two ethnic groups were not significantly different in cytochrome-b(5) concentrations but were significantly different in total CYP concentrations and NADPH-P450 reductase activity (P < 0.05). Significant ethnic differences in intrinsic clearance were observed for CYP1A2, CYP2C9, CYP2C19 and CYP2E1; the median values of the Chinese group were 54, 58, 26, and 35% of the corresponding values of the Caucasian group, respectively. These differences were associated with differences in Michaelis constant or maximum velocity. Despite negligible difference in intrinsic clearance, the Michaelis constant of CYP2B6 appeared to have a significant ethnic difference. No ethnic difference was observed for CYP2A6, CYP2C8, CYP2D6 and CYP3A.ConclusionsThese data extend our knowledge on the ethnic differences in CYP enzymes and will have implications for drug discovery and drug therapy for patients from different ethnic origins.

| S-EPMC3269586 | biostudies-literature

Oxidation of N-Nitrosoalkylamines by human cytochrome P450 2A6: sequential oxidation to aldehydes and carboxylic acids and analysis of reaction steps.

Project description:Cytochrome P450 (P450) 2A6 activates nitrosamines, including N,N-dimethylnitrosamine (DMN) and N,N-diethylnitrosamine (DEN), to alkyl diazohydroxides (which are DNA-alkylating agents) and also aldehydes (HCHO from DMN and CH(3)CHO from DEN). The N-dealkylation of DMN had a high intrinsic kinetic deuterium isotope effect ((D)k(app) approximately 10), which was highly expressed in a variety of competitive and non-competitive experiments. The (D)k(app) for DEN was approximately 3 and not expressed in non-competitive experiments. DMN and DEN were also oxidized to HCO(2)H and CH(3)CO(2)H, respectively. In neither case was a lag observed, which was unexpected considering the k(cat) and K(m) parameters measured for oxidation of DMN and DEN to the aldehydes and for oxidation of the aldehydes to the carboxylic acids. Spectral analysis did not indicate strong affinity of the aldehydes for P450 2A6, but pulse-chase experiments showed only limited exchange with added (unlabeled) aldehydes in the oxidations of DMN and DEN to carboxylic acids. Substoichiometric kinetic bursts were observed in the pre-steady-state oxidations of DMN and DEN to aldehydes. A minimal kinetic model was developed that was consistent with all of the observed phenomena and involves a conformational change of P450 2A6 following substrate binding, equilibrium of the P450-substrate complex with a non-productive form, and oxidation of the aldehydes to carboxylic acids in a process that avoids relaxation of the conformation following the first oxidation (i.e. of DMN or DEN to an aldehyde).

| S-EPMC2832954 | biostudies-literature

Roles of Human Liver Cytochrome P450 Enzymes in Tenatoprazole Metabolism.

Project description:Tenatoprazole, a newly developed proton pump inhibitor candidate, was developed as an acid inhibitor for gastric acid hypersecretion disorders such as gastric ulcer and reflux esophagitis. It is known that tenatoprazole is metabolized to three major metabolites of 5'-hydroxy tenatoprazole, tenatoprazole sulfide, and tenatoprazole sulfone in human liver, primarily catalyzed by CYPs 2C19 and 3A4. While CYP2C19 prefers the hydroxylation of tenatoprazole at C-5' position, CYP3A4 is mainly involved in sulfoxidation reaction to make tenatoprazole sulfone. Tenatoprazole sulfide is a major human metabolite of tenatoprazole and is formed spontaneously and non-enzymatically from tenatoprazole. However, its metabolic fate in the human liver is not fully known. Furthermore, no systematic metabolic study has been performed to study tenatoprazole or tenatoprazole sulfide. Here, we studied the functions of human cytochromes P450 in the metabolic pathway of tenatoprazole and tenatoprazole sulfide by using recombinant human P450s and human liver microsomes. Both CYP 2C19 and CYP3A4 showed distinct regioselective and stereospecific monooxygenation activities toward tenatoprazole and tenatoprazole sulfide. Furthermore, a new major metabolite of tenatoprazole sulfide was found, 1'-N-oxy-5'-hydroxytenatoprzole sulfide, which has never been reported. In conclusion, the metabolic fates of tenatoprazole and tenatoprazole sulfide should be considered in the clinical use of tenatoprazole.

| S-EPMC9863764 | biostudies-literature

Characterization of the metabolism of fenretinide by human liver microsomes, cytochrome P450 enzymes and UDP-glucuronosyltransferases.

Project description:Background and purposeFenretinide (4-HPR) is a retinoic acid analogue, currently used in clinical trials in oncology. Metabolism of 4-HPR is of particular interest due to production of the active metabolite 4'-oxo 4-HPR and the clinical challenge of obtaining consistent 4-HPR plasma concentrations in patients. Here, we assessed the enzymes involved in various 4-HPR metabolic pathways.Experimental approachEnzymes involved in 4-HPR metabolism were characterized using human liver microsomes (HLM), supersomes over-expressing individual human cytochrome P450s (CYPs), uridine 5'-diphospho-glucoronosyl transferases (UGTs) and CYP2C8 variants expressed in Escherichia coli. Samples were analysed by high-performance liquid chromatography and liquid chromatography/mass spectrometry assays and kinetic parameters for metabolite formation determined. Incubations were also carried out with inhibitors of CYPs and methylation enzymes.Key resultsHLM were found to predominantly produce 4'-oxo 4-HPR, with an additional polar metabolite, 4'-hydroxy 4-HPR (4'-OH 4-HPR), produced by individual CYPs. CYPs 2C8, 3A4 and 3A5 were found to metabolize 4-HPR, with metabolite formation prevented by inhibitors of CYP3A4 and CYP2C8. Differences in metabolism to 4'-OH 4-HPR were observed with 2C8 variants, CYP2C8*4 exhibited a significantly lower V(max) value compared with *1. Conversely, a significantly higher V(max) value for CYP2C8*4 versus *1 was observed in terms of 4'-oxo formation. In terms of 4-HPR glucuronidation, UGTs 1A1, 1A3 and 1A6 produced the 4-HPR glucuronide metabolite.Conclusions and implicationsThe enzymes involved in 4-HPR metabolism have been characterized. The CYP2C8 isoform was found to have a significant effect on oxidative metabolism and may be of clinical relevance.

| S-EPMC3042207 | biostudies-literature

Exploring the Interactome of Cytochrome P450 2E1 in Human Liver Microsomes with Chemical Crosslinking Mass Spectrometry.

Project description:Aiming to elucidate the system-wide effects of the alcohol-induced increase in the content of cytochrome P450 2E1 (CYP2E1) on drug metabolism, we explored the array of its protein-protein interactions (interactome) in human liver microsomes (HLM) with chemical crosslinking mass spectrometry (CXMS). Our strategy employs membrane incorporation of purified CYP2E1 modified with photoreactive crosslinkers benzophenone-4-maleimide and 4-(N-succinimidylcarboxy)benzophenone. Exposure of bait-incorporated HLM samples to light was followed by isolating the His-tagged bait protein and its crosslinked aggregates on Ni-NTA agarose. Analyzing the individual bands of SDS-PAGE slabs of thereby isolated protein with the toolset of untargeted proteomics, we detected the crosslinked dimeric and trimeric complexes of CYP2E1 with other drug-metabolizing enzymes. Among the most extensively crosslinked partners of CYP2E1 are the cytochromes P450 2A6, 2C8, 3A4, 4A11, and 4F2, UDP-glucuronosyltransferases (UGTs) 1A and 2B, fatty aldehyde dehydrogenase (ALDH3A2), epoxide hydrolase 1 (EPHX1), disulfide oxidase 1α (ERO1L), and ribophorin II (RPN2). These results demonstrate the exploratory power of the proposed CXMS strategy and corroborate the concept of tight functional integration in the human drug-metabolizing ensemble through protein-protein interactions of the constituting enzymes.

| S-EPMC8869672 | biostudies-literature

Structural insight into the altered substrate specificity of human cytochrome P450 2A6 mutants.

Project description:Human P450 2A6 displays a small active site that is well adapted for the oxidation of small planar substrates. Mutagenesis of CYP2A6 resulted in an increased catalytic efficiency for indole biotransformation to pigments and conferred a capacity to oxidize substituted indoles (Wu, Z.-L., Podust, L.M., Guengerich, F.P. J. Biol. Chem. 49 (2005) 41090-41100.). Here, we describe the structural basis that underlies the altered metabolic profile of three mutant enzymes, P450 2A6 N297Q, L240C/N297Q and N297Q/I300V. The Asn297 substitution abolishes a potential hydrogen bonding interaction with substrates in the active site, and replaces a structural water molecule between the helix B'-C region and helix I while maintaining structural hydrogen bonding interactions. The structures of the P450 2A6 N297Q/L240C and N297Q/I300V mutants provide clues as to how the protein can adapt to fit the larger substituted indoles in the active site, and enable a comparison with other P450 family 2 enzymes for which the residue at the equivalent position was seen to function in isozyme specificity, structural integrity and protein flexibility.

| S-EPMC2773796 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data