Oxidative Metabolism of Estrone Modified by Genistein and Bisphenol A in Rat Liver Microsomes

Genistein, the main isoflavone from soy, and bisphenol A （BPA）, a food contaminant, are considered ubiquitous xenoestrogens. Here we investigated the influence of genistein and BPA on estrone （El） metabolism in rat liver microsomes. Both substances inhibited the 2-hydroxylation and 16a-hydroxylation of E1, but in different degrees, thereby reducing the 2-OH-E1/16a-OH-E1 ratio,

Reductive Metabolism of Nitroaromatic Compounds by Various Liver Microsomes

Nitroaromatic compounds were reductively metabolized to the corresponding amine compounds via the intermediate hydroxylamines by liver microsomes from pig,rat,chook,cattle,sheep,paralichthys olivaceus and cyprinoid in varied reactivity.Similar with baker＇s yeast,the pig,rat and sheep liver microsomes exhibited high reactivity toward 4-nitro-1,2-dicyanbenzen（1a）,while the cyprinoid liver microsomes were inefficient.Contrasted to compound 1a,monocyannitrobenzene（2a） was difficult to reduce by pig liver microsomes.In opposition to grape cells,pig liver microsomes exhibited activities toward some aromatic hydroxylamine compounds.

Flavonoids Reduce Lipid Peroxides and Increase Glutathione Levels in Pooled Human Liver Microsomes (HLMs)

The effects of each of the flavonoids;genistein (G), quercetin (Q) and kaempferol (K) at several doses on lipid peroxides (LP) and reduced glutathione (GSH) in pooled human liver microsomes (HLMs) were investigated following the oxidative damage for 4, 6, 18 and 24 hr. HLMs (1 mg/ml) were exposed to each of the above flavonoids at 0, 5, 10, 15, 20 or 25 μM and incubated for the respective times as previously stated. Our hypothesis was that HLMs exposed to the flavonoids for the respective exposure times can decrease LP and increase GSH in HLMs to better cope with the oxidative stress. The results of our studies indicate that each of the flavonoids significantly (p < 0.01) decreased LP compared to their respective controls. The highest decrease in LP was observed for K followed by Q and G. Significant increases (p < 0.01) in GSH were observed for the flavonoid doses tested with the highest levels observed for Q for the 24-hr. incubation. The findings suggest that the flavonoids modulate oxidative stress in HLMs by decreasing LP and such decreases in LPs may be due to the increasing and or the replenished levels of GSH in the said cells to better cope with the oxidative stress.

Inhibition of Re Du Ning Injection on Enzyme Activities of Rat Liver Microsomes Using Cocktail Method

Objective Re Du Ning Injection （RDN）, a Chinese materia medica injection, is made from the extracts of LoniceraeJaponicae Flos, Gardeniae Fructus, and Artemisiae Annuae Herba. Since last decade, RDN has been widely used in China for the treatment of viral infection, fever, and inflammation. To assess the potential interacting of RDN with co-administered drugs, the inhibitory effects of RDN on the enzyme activities （CYP1A1, CYP1A2, CYP2C11, CYP2D1, and CYP3A1/2） of rat liver microsomes were investigated by a cocktail method. Methods A sensitive and specific LC-MS method capable of simultaneous quantification of five metabolites in rat liver microsomes was developed and validated. Then RDN （0.625%-1.0%） was incubated with rat liver microsomes and specific substrates. The enzyme activities were expressed as the formation rate of the specific metabolites of the substrates （pmol- mg. protein-1 . min-1）. Results RDN competitively inhibited the activities of CYP1A2 and CYP2C11, with inhibition constant （/~） values determined to be 0.18% and 0.63%, respectively. RDN exhibited the mixed inhibition on the activity of CYP2D1, with a K1 value of 0.15%. The activities of CYP1A1 and CYP3A1/2 were not markedly inhibited even by 1.0% RDN. Conclusion RDN could inhibit the rat enzyme activities of CYP1A2, 2Cll, and 2D1 in vitro with different inhibition modes, which is worthy of promoting safety and efficacy of RDN.

Inhibition of Magnolol and Honokiol on Cytochrome P450 Enzymes in Rat and Human Liver Microsomes

Objective The purpose of this work is to evaluate the in vitro inhibitory effect of magnolol（MN） and honokiol（HN） on rat / human cytochrome P450（CYP） enzymes（1A2/1A2, 2D/2D6, 3A/3A4, 2E1/2E1, and 2C/2C9）. Methods Rat liver microsomes（RLM） and human liver microsomes（HLM） were used as the enzyme sources. After the probe substrate of each CYP isoforms was co-incubated individually with MN or HN in RLM or HLM, the metabolite production of each probe substrate in RLM and HLM incubation medium was determined and used to evaluate the activity of corresponding CYP isoforms. Results MN inhibited rat CYP1A2 and human CYP3A4 with the IC50 values of 10.0 and 56.2 μmol/L, respectively. HN inhibited rat CYP1A2 and CYP2E1, human CYP1A2 and CYP3A4 with the IC50 values of 12.1, 12.6, 17.8, and 43.9 μmol/L, respectively. Conclusion HN is a moderate or weak inhibitor of human CYP1A2. Both MN and HN are weak or non inhibitors of the other tested human CYP isoforms. The results suggest that no significant metabolic interaction seems likely to occur when the substrate drugs of CYP isoforms tested in the present work are co-administered with MN and HN.

Inhibition of Cytochrome P450 by Nomilin and Obacunone and Potential Mechanism in Human Liver Microsomes

Objective Nomilin and obacunone are two important limonoids that are well known for their anticancer effect. Previous studies showed that limonoids had inhibitory effect on cytochrome P450 3A4（CYP3A4）. However these effects are inconclusive with regards to prediction of potential drug interactions. Methods Nomilin or obacunone was pre-incubated with HLMs for 30 min. Following 10-fold dilution from the pre-incubation concentration, a second incubation was performed in the presence of NADPH and cytochrome P450 substrates for 15 min. The reaction was quenched and the supernatants were analyzed by chromatography/mass spectrometry. Results In this study, nomilin and obacunone showed potent inhibitory effect on CYP3A4 with the IC_（50） values of 3.50 and 6.08 μmol/L, respectively. The inhibition of CYP3A4 was in a time-, concentration-and NADPH-dependent manner with Ki values of 2.92 and 1.25 μmol/L and Kinact values of 0.033 and 0.078 min^（-1） for nomilin and obacunone respectively. These results elucidated that they were time-dependent inhibitors for CYP3A4. Conclusion Concomitant use of limonoids and other drugs may call for extra caution for purposes of clinical safety.

An Integrated Study for the Utilization of Anthraquinone Compounds Extract“Heshouwu”In vivo and their Comparative Metabolism in Liver Microsomes Using UPLC-ESI-Q-TOF/MS^(n)

Objective: Anthraquinone(AQ), a major bioactive component of the traditional Chinese medicine He ShouW u, has widespread applications in industry and medicine. The objective of the current study is to explore the differences in the bioavailability of anthraquinones in vivo and the metabolism in liver microsomes. Materials and Methods: In vivo, we used a reliable UPLC?ESI?Qq Q?MS/MS method to measure seven AQ compounds in the jugular vein plasma of rats following oral administration of He Shou Wu. Furthermore, in order to quantify the bioavailability of AQs in vivo and to further understand the metabolism of these compounds, we compared the in vitro metabolism of AQ in different species with respect to metabolic profiles, the enzymes involved, and catalytic efficiency using liver microsomes from human(HLM), mouse(MLM), rat(RLM), and beagle dog(DLM). Results: We identified two metabolic pathways, including the hydroxylation and glucuronidation of AQ, in the liver microsomes of humans and other species using UPLC?ESI?Q?TOF. We found that substitutions on the AQ ring were crucial to the activity and regioselectivity of its hydroxylation. In general, hydroxylation activity decreased greatly with β?COOH(rhein) and enhanced dramatically with β?OH(emodin). We also found that glucuronidation of the compound emodin?8?O?β?D?glucoside acts as the main isoform in AQ hydroxylation in HLM and DLM. Total microsomal intrinsic clearance values for AQ were greatest in mouse microsomes, followed by those in dog, human, and rat microsomes. Conclusion: The absorption of different anthrquinone compounds varied based on the compound structure, the metabolism types and products of anthraquinones in liver microsomes were different in different species. These findings provide vital information for a deeper unuunderstanding of the metabolism of AQs.

Electrochemical and in silico approaches for liver metabolic oxidation of antitumor-active triazoloacridinone C-1305

5-Dimethylaminopropylamino-8-hydroxytriazoloacridinone(C-1305)is a promising antitumor compound developed in our laboratory.A better understanding of its metabolic transformations is still needed to explain the multidirectional mechanism of pharmacological action of triazoloacridinone derivatives at all.Thus,the aim of the current work was to predict oxidative pathways of C-1305 that would reflect its phase I metabolism.The multi-tool analysis of C-1305 metabolism included electrochemical conversion and in silico sites of metabolism predictions in relation to liver microsomal model.In the framework of the first approach,an electrochemical cell was coupled on-line to an electrospray ionization mass spectrometer.The effluent of the electrochemical cell was also injected onto a liquid chromatography column for the separation of different products formed prior to mass spectrometry analysis.In silico studies were performed using MetaSite software.Standard microsomal incubation was employed as a reference procedure.We found that C-1305 underwent electrochemical oxidation primarily on the dialkylaminoalkylamino moiety.An unknown N-dealkylated and hydroxylated C-1305 products have been identified.The electrochemical system was also able to simulate oxygenation reactions.Similar pattern of C-1305 metabolism has been predicted using in silico approach.Both proposed strategies showed high agreement in relation to the generated metabolic products of C-1305.Thus,we conclude that they can be considered as simple alternatives to enzymatic assays,affording time and cost efficiency.

Determination of Synthetic Impurities and Metabolic Products of F-ADAM, a Positron Emission Tomography Imaging Agent for Serotonin Transporter (SERT) Using HPLC-Tandem Mass Spectrometry

A high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) analytical method was developed to determine the identity of impurities resulting from the synthesis of N,N-dimethyl-2-(2-amino-4-fluorophenylthio)benzyl-amine (F-ADAM), as well as its metabolic products by rat liver microsomes. 18F-ADAM is an important positive electron emission ligand commonly employed as a radio-imaging agent for serotonin transporter (SERT) in the brain. F-ADAM and its derivatives were separated using HPLC on a C4-phenyl column with an ammonium formate aqueous buffer/acetonitrile programmed mobile phase. Synthetic contaminants and metabolic products were identified using fragmentation spectra obtained by tandem mass spectrometry. We show that F-ADAM is unstable in methanol, and propose the use of acetonitrile to generate optimal chromatogram. A Cl-substituted species was found to be the major impurity resulting from the F-ADAM synthetic process. The metabolic products of F-ADAM by rat liver microsomes were characterized by oxidization of the sulfur moiety to sulfoxide, demethylation of the dimethylamine moiety, and oxidative defluorination/deamination. These results elucidate the by-products of F-ADAM synthetic and metabolic processes, and provide direction for the application of this imaging agent to biosystems properly.

The metabolism and hepatotoxicity of ginkgolic acid(17:1) in vitro

Pharmacological activities and adverse side effects of ginkgolic acids(GAs), major components in extracts from the leaves and seed coats of Ginkgo biloba L, have been intensively studied. However, there are few reports on their hepatotoxicity. In the present study, the metabolism and hepatotoxicity of GA(17:1), one of the most abundant components of GAs, were investigated. Kinetic analysis indicated that human and rat liver microsomes shared similar metabolic characteristics of GA(17:1) in phase I and II metabolisms. The drug-metabolizing enzymes involved in GA(17:1) metabolism were human CYP1 A2, CYP3 A4, UGT1 A6, UGT1 A9, and UGT2 B15, which were confirmed with an inhibition study of human liver microsomes and recombinant enzymes. The MTT assays indicated that the cytotoxicity of GA(17:1) in HepG2 cells occurred in a time-and dose-dependent manner. Further investigation showed that GA(17:1) had less cytotoxicity in primary rat hepatocytes than in HepG2 cells and that the toxicity was enhanced through CYP1 A-and CYP3 A-mediated metabolism.

In vitro Metabolism of Strychnine by Human Cytochrome P450 and Its Interaction with Glycyrrhetic Acid

Objective To investigate the metabolism of strychnine(STN)and the metabolic interaction between STN and glycyrrhetic acid(GA)in vitro.Methods Human liver microsomes(HLM)and human recombinant cytochrome P450(CYP)isoforms were employed to study the metabolism of STN and the metabolic interaction of STN with GA in vitro.Results In HLM,the Km,Vmax,and clearance of STN were 88.50μmol/L,0.88 nmol/(mg·min),and 9.93 mL/(mg·min),respectively.STN was metabolized mainly by CYP3A4.However,STN noncompetitively inhibited CYP3A4-catalyzed testosterone 6β-hydroxylation with IC50 value of 5.9μmol/L and Ki value of 5.5 μmol/L.Moreover,GA competitively inhibited STN metabolism with IC50 value of 10.6μmol/L and Ki value of 17.7μmol/L.Conclusion Although STN is mainly metabolized by CYP3A4 in vitro,STN has noncompetitive inhibition on CYP3A4-catalyzed testosterone 6β-hydroxylation.Moreover,GA could competitively inhibit STN metabolism.The present work is helpful to elucidate the metabolic interaction between STN and GA.