Microsomal Suspensions Research Articles

Synthesis and in vitro Metabolic Stability of Sterically Shielded Antimycobacterial Phenylalanine Amides.

Nα-aroyl-N-aryl-phenylalanine amides (AAPs) are RNA polymerase inhibitors with activity against Mycobacterium tuberculosis and non-tuberculous mycobacteria. We observed that AAPs rapidly degrade in microsomal suspensions, suggesting that avoiding hepatic metabolism is critical for their effectiveness in vivo. As both amide bonds are potential metabolic weak points of the molecule, we synthesized 16 novel AAP analogs in which the amide bonds are shielded by methyl or fluoro substituents in close proximity. Some derivatives show improved microsomal stability, while being plasma-stable and non-cytotoxic. In parallel with the metabolic stability studies, the antimycobacterial activity of the AAPs against Mycobacterium tuberculosis, Mycobacterium abscessus, Mycobacterium avium and Mycobacterium intracellulare was determined. The stability data are discussed in relation to the antimycobacterial activity of the panel of compounds and reveal that the concept of steric shielding of the anilide groups by a fluoro substituent has the potential to improve the stability and bioavailability of AAPs.

In vitro assessment of competitive and time-dependent inhibition of the nevirapine metabolism by nortriptyline in rats

Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor of human immunodeficiency virus type 1 (HIV-1) widely used as a component of High Active Antiretroviral Therapy (HAART) since it is inexpensive, readily absorbed after oral administration and non-teratogenic. In the present work, the mechanism of a previously described pharmacokinetic interaction between NVP and the antidepressant drug nortriptyline (NT) was studied using rat hepatic microsomes. The obtained results showed a competitive inhibition of the NVP metabolism by NT. The three main NVP metabolites (2-OH-NVP, 3-OH-NVP and 12-OH-NVP) where competitively inhibited with similar inhibitory constant values (Ki = 4.01, 3.97 and 4.40 μM, respectively). Time-dependent inhibition of the NVP metabolism was also detected, with a 2.5-fold reduction in the IC50 values of NT for 2-, 3-, and 12-OH-NVP formation when NT was preincubated with the microsomal suspension in the presence of an NADPH-generating system. A concentration-dependent inhibition of the formation of NVP metabolites by the main NT metabolite (10-OH-NT) was also observed, however, the inhibitory potency of 10-OH-NT was much lower than that of the parent drug. The apparent hepatic intrinsic clearance of NVP determined in these in vitro experiments was used to predict the in vivo clearance of NVP using the “well-stirred” and the “parallel-tube” models, resulting in values close to those previously observed in vivo clearance. Finally, a good prediction of the increase in the plasma concentrations of NVP when co-administered with NT was obtained employing the inhibitory constant of NT determined in vitro and the estimated plasma concentration of NT entering the liver.

In vitro pharmacokinetic profile of a benzopyridooxathiazepine derivative using rat microsomes and hepatocytes: Identification of phases I and II metabolites

In the present study, the in vitro metabolic behavior of a benzopyridooxathiazepine (BZN), a potent tubulin polymerization inhibitor, was investigated by liquid chromatography-UV detection (LC-UV). First, simple and fast LC-UV methods have been optimized and validated to evaluate the pharmacokinetic profile of BZN using rat liver microsomes or hepatocytes primary cultures suspensions. Whatever the medium investigated, baseline resolution between the internal standard and BZN was achieved in a run time less than 15min using a Symmetry ODS column (150mm×4.6mm i.d., 5μm) and a mobile phase consisting of acetonitrile/water/formic acid 60:40:0.1 (v/v/v). Linearity was assessed in the 0.1–50μM and in the 0.05–5μM concentration ranges, respectively, in microsomal and hepatocyte matrix. According to the novel strategy based on the build of the accuracy profile, total error of the developed methods was included within the ±10% limits of acceptance. Then, from incubation of BZN with both liver microsomes and or hepatocytes, structural informations on phase I and phase II metabolites were acquired using liquid chromatography coupled to electrospray orbitrap mass spectrometer (LC–MS). Mass spectrum, double bond equivalent and elemental composition were useful data to access to the chemical structure of each metabolite. In microsomal suspension, four main metabolites were observed including monohydroxylation and dihydroxylation of the benzopyridooxathiazepine core, demethylation of the methoxyphenyl moiety, as well as their combinations. The phase II metabolites detected in hepatocytes suspension were the glucuronide adducts of both demethylated BZN and mono-oxygenated BZN. Based on the structural elucidation of the metabolites detected, we proposed an in vitro metabolic pathway of BZN, a new tubulin polymerization inhibitor.

Effects of orally applied butyrate bolus on histone acetylation and cytochrome P450 enzyme activity in the liver of chicken – a randomized controlled trial

BackgroundButyrate is known as histone deacetylase inhibitor, inducing histone hyperacetylation in vitro and playing a predominant role in the epigenetic regulation of gene expression and cell function. We hypothesized that butyrate, endogenously produced by intestinal microbial fermentation or applied as a nutritional supplement, might cause similar in vivo modifications in the chromatin structure of the hepatocytes, influencing the expression of certain genes and therefore modifying the activity of hepatic microsomal drug-metabolizing cytochrome P450 (CYP) enzymes.MethodsAn animal study was carried out in chicken as a model to investigate the molecular mechanisms of butyrate’s epigenetic actions in the liver. Broiler chicks in the early post-hatch period were treated once daily with orally administered bolus of butyrate following overnight starvation with two different doses (0.25 or 1.25 g/kg body weight per day) for five days. After slaughtering, cell nucleus and microsomal fractions were separated by differential centrifugation from the livers. Histones were isolated from cell nuclei and acetylation of hepatic core histones was screened by western blotting. The activity of CYP2H and CYP3A37, enzymes involved in biotransformation in chicken, was detected by aminopyrine N-demethylation and aniline-hydroxylation assays from the microsomal suspensions.ResultsOrally added butyrate, applied in bolus, had a remarkable impact on nucleosome structure of hepatocytes: independently of the dose, butyrate caused hyperacetylation of histone H2A, but no changes were monitored in the acetylation state of H2B. Intensive hyperacetylation of H3 was induced by the higher administered dose, while the lower dose tended to increase acetylation ratio of H4. In spite of the observed modification in histone acetylation, no significant changes were observed in the hepatic microsomal CYP2H and CYP3A37 activity.ConclusionOrally added butyrate in bolus could cause in vivo hyperacetylation of the hepatic core histones, providing modifications in the epigenetic regulation of cell function. However, these changes did not result in alteration of drug-metabolizing hepatic CYP2H and CYP3A37 enzymes, so there might be no relevant pharmacoepigenetic influences of oral application of butyrate under physiological conditions.

Failure of Prion Protein Oxidative Folding Guides the Formation of Toxic Transmembrane Forms

The mechanism by which pathogenic mutations in the globular domain of the cellular prion protein (PrP(C)) increase the likelihood of misfolding and predispose to diseases is not yet known. Differences in the evidences provided by structural and metabolic studies of these mutants suggest that in vivo folding could be playing an essential role in their pathogenesis. To address this role, here we use the single or combined M206S and M213S artificial mutants causing labile folds and express them in cells. We find that these mutants are highly toxic, fold as transmembrane PrP, and lack the intramolecular disulfide bond. When the mutations are placed in a chain with impeded transmembrane PrP formation, toxicity is rescued. These results suggest that oxidative folding impairment, as on aging, can be fundamental for the genesis of intracellular neurotoxic intermediates key in prion neurodegenerations.

In vitro determination of the CYP 3A4 activity in rat hepatic microsomes by liquid-phase extraction and HPLC-photodiode array detection

IntroductionCYP3A4 is one of the most important of all drug-metabolizing enzymes. Although several direct or indirect quantification methods have been proposed for the determination of the CYP3A4 activity, the sample preparation is mostly tedious and usually requires time consuming separate extraction steps and solid-phase extraction. MethodsHere, we developed a simple and selective HPLC method, coupled to photodiode array detection for direct determination of CYP3A4-mediated testosterone hydroxylase activity in hepatic microsomes of rats. After microsome incubation, a single-step liquid‐phase extraction of specific substrates, testosterone and its metabolite 6-hydroxytestosterone, together with the salicylamid acid used as an internal standard, was applied. The analytical method was fully validated by the determination of different parameters (intra‐ and inter‐day variability of 6-β-OH-testosterone concentration, accuracy and limit of detection). Finally, this method was applied to quantify the CYP3A4 activity of rats intravenously administered with either the mesoporous iron(III) trimesate MIL-100 nanocarrier (MIL stands for Materials from Institut Lavoisier) or with its corresponding organic linker. ResultsAll analytes were simultaneously separated from a single run shorter than 10min, reaching relative standard deviations of intra- and inter-day precision <18.5% and an accuracy of estimated 6-β-OH-testosterone concentrations ranging from 95 to 111%. The mean±standard deviation absolute recoveries of 6-β-OH-testosterone at 0.01, 1.00 and 20.00μg/mL were 97±4%, 101±3% and 99±2%, respectively while it reached 98±3% for the internal standard. DiscussionThe developed HPLC-PDA method enables the accurate and sensitive determination of the CYP3A4 activity through the quantification of the 6-β-OH-testosterone produced by the CYP3A4-mediated testosterone hydroxylase in rat hepatic microsomal suspensions. Additionally, the rapid procedure offers an economical advantage with respect to resources and operator time. Finally, the determination of CYP3A4 activity in hepatic microsomes of rats administered with nanoparticles of the porous iron(III) trimesate nanocarrier shows no significant differences between control, trimesic and nanoparticle groups, evidencing that the linker is not metabolized by CYP3A4.

Interactions between Hemoglobin and Cod Muscle Constituents following Treatment at Extreme pH Values

Acid and/or alkaline solubilization is a recent method developed to separate proteins from muscle foods with good functional properties. However, exposure of the muscle and its components to low pH values has been shown to promote lipid oxidation, limiting therefore the applications of this novel method. This research aimed primarily to study the physicochemical changes of the fish membranes brought about during acid or alkali solubilization processes. The effect on lipid oxidation and the possible role of the water soluble fraction of the muscle (press juice) as a potent antioxidant were also investigated. Model systems comprising minced cod muscle or cod microsomal suspensions were used. Results showed that acid or alkaline treatment (pH < 3.5 or pH > 10.5) of cod membranes significantly delayed lipid oxidation. Added triacylglycerols to washed cod system treated at low pH did not enhance hemoglobin-mediated lipid oxidation. Decreased precipitation of hemoglobin was observed with the alkali-treated membranes at all protein concentrations compared to the acid-treated and the untreated membranes. Finally, the addition of press juice to washed cod muscle tissue or to the membrane model system, significantly delayed hemoglobin lipid oxidation. The results of this study can be used to improve pH-shifting technologies to avoid or decrease lipid oxidation problems. Also, the use of press-juice from cod muscle as means of protecting the muscle against lipid oxidation is suggested.

Kinetics of ethylene and ethylene oxide in subcellular fractions of lungs and livers of male B6C3F1 mice and male fischer 344 rats and of human livers.

Ethylene (ET) is metabolized in mammals to the carcinogenic ethylene oxide (EO). Although both gases are of high industrial relevance, only limited data exist on the toxicokinetics of ET in mice and of EO in humans. Metabolism of ET is related to cytochrome P450-dependent mono-oxygenase (CYP) and of EO to epoxide hydrolase (EH) and glutathione S-transferase (GST). Kinetics of ET metabolism to EO and of elimination of EO were investigated in headspace vessels containing incubations of subcellular fractions of mouse, rat, or human liver or of mouse or rat lung. CYP-associated metabolism of ET and GST-related metabolism of EO were found in microsomes and cytosol, respectively, of each species. EH-related metabolism of EO was not detectable in hepatic microsomes of rats and mice but obeyed saturation kinetics in hepatic microsomes of humans. In ET-exposed liver microsomes, metabolism of ET to EO followed Michaelis-Menten-like kinetics. Mean values of Vmax [nmol/(min·mg protein)] and of the apparent Michaelis constant (Km [mmol/l ET in microsomal suspension]) were 0.567 and 0.0093 (mouse), 0.401 and 0.031 (rat), and 0.219 and 0.013 (human). In lung microsomes, Vmax values were 0.073 (mouse) and 0.055 (rat). During ET exposure, the rate of EO production decreased rapidly. By modeling a suicide inhibition mechanism, rate constants for CYP-mediated catalysis and CYP inactivation were estimated. In liver cytosol, mean GST activities to EO expressed as Vmax/Km [μl/(min·mg protein)] were 27.90 (mouse), 5.30 (rat), and 1.14 (human). The parameters are most relevant for reducing uncertainties in the risk assessment of ET and EO.

Characterization of [6]-gingerol metabolism in rat by liquid chromatography electrospray tandem mass spectrometry

[6]-Gingerol is a structural analog of capsaicin, an agonist of the transient receptor potential channel vanilloid 1, which is known to have therapeutic properties for the treatment of pain and inflammation. A selective and sensitive quantitative method for the determination of [6]-gingerol by HPLC-ESI/MS/MS was developed. The method consisted of a protein precipitation extraction followed by analysis using liquid chromatography electrospray tandem mass spectrometry. The chromatographic separation was achieved using a Thermo 100 × 2.1 mm C(8) column combined with an isocratic mobile phase composed of acetonitrile, water and formic acid (80:20:0.1) at a flow rate of 250 μL/min. The mass spectrometer was operating in SRM mode and an analytical range set at 20-5000 ng/mL was used to construct a calibration curve in rat plasma. The interbatch precision (%CV) and accuracy (%NOM) observed were 2.9-10.8% and 98.1-102.1% in rat plasma. Similarly, precision and accuracy in rat liver microsomal suspension were also evaluated at nominal concentrations of 1, 25 and 100 μm; the precision (%CV) was <3.4% and the accuracy (%NOM) observed ranged from 89.7 to 109.4%. An in vitro metabolic stability study using rat liver microsomes was performed to determine intrinsic clearance of [6]-gingerol. The results show slow degradation with a T(1/2) of 163 min and relatively low intrinsic clearance suggesting that phase I metabolism may not be a major contributor of the drug clearance. Further analyses were performed to characterize in vitro and in vivo metabolites. Three main phase I metabolites and four phase II metabolites were identified by HPLC-MS/MS and HPLC-MSD TOF. However, the results suggest that glucuronidation of hydroxylated [6]-gingerol is the primary metabolite excreted in rat urine.

Metabolic stability and determination of cytochrome P450 isoenzymes' contribution to the metabolism of medetomidine in dog liver microsomes

Medetomidine is a potent and selective alpha2-adrenergic agonist. The activation of alpha2-adrenergic receptor mediates a variety of effects including sedation, analgesia, relief of anxiety, vasoconstriction and bradycardia. However, our main interest is the sedative effects of medetomidine when used as a premedicant prior surgery in companion animals, especially in dogs. Recently, data suggested that following intravenous infusion at six dosing regiments non-linear pharmacokinetics was observed. Major causes of non-linear pharmacokinetics are the elimination of the drug not following a simple first-order kinetics and/or the elimination half-life changing due to saturation of an enzyme system. The goal of this study was to establish the metabolic stability and determine the metabolic pathway of medetomidine in dog liver microsomes. Consequently, Michaelis-Menten parameters (V(max), K(m)), T(1/2) and CL(i) were determined. The incubations were performed in a microcentrifuge tube and containing various concentrations of medetomidine (10-5000 nM), 1 mg/mL of microsomal proteins suspended in 0.1 M phosphate buffer, pH 7.4. Microsomal suspensions were preincubated with NADPH (1 mM) for 5 min at 37 degrees C prior to fortification with medetomidine. Samples were taken at various time points for kinetic information and the initial velocity (v(i)) was determined after 10 min incubation. The reaction was stopped by the addition of an internal standard solution (100 ng/mL of dextrometorphan in acetone). Medetomidine concentrations were determined using a selective and sensitive HPLC-ESI/MS/MS method. Using non-linear regression, we determined a K(m) value of 577 nM, indicating relatively low threshold enzyme saturation consistent with previous in vivo observation. The metabolic stability was determined at a concentration of 100 nm (<<K(m)) and the observed T(1/2) was 90 min with a CL(i) of 0.008 mL/min indicating moderately low clearance in dog liver microsomes, also consistent with previous in vivo data. Moreover, results suggest that principally medetomidine is metabolized by the CYP3A with a small contribution from CYP2D and CYP2E. The participation of CYP3A is an important discovery since medetomidine is used as a premedicant in combination with fentanyl, ketamine and/or midazolam. These findings combined with a low K(m) value may indicate that medetomidine can competitively inhibit the metabolism of these drugs and consequently significantly impair metabolic clearance.

Identification of a Radical Formed in the Reaction Mixtures of Ram Seminal Vesicle Microsomes with Arachidonic Acid Using High Performance Liquid Chromatography-Electron Spin Resonance Spectrometry and High Performance Liquid Chromatography-Electron Spin Resonance-Mass Spectrometry

The reaction of ram seminal vesicle (RSV) microsomes with arachidonic acid (AA) was examined using electron spin resonance (ESR), high performance liquid chromatography-electron spin resonance spectrometry (HPLC-ESR), and high performance liquid chromatography-electron spin resonance-mass spectrometry (HPLC-ESR-MS) combined use of spin trapping technique. A prominent ESR spectrum (αN = 1.58 mT and αHβ = 0.26 mT) was observed in the complete reaction mixture of ram seminal vesicle microsomes with arachidonic acid containing 2.0 mg protein/ml ram seminal vesicle (RSV) microsomal suspension, 0.8 mM arachidonic acid, 0.1 M 4-POBN, and 24 mM tris/HCl buffer (pH 7.4). The ESR spectrum was hardly observed for the complete reaction mixture without the RSV microsomes. The formation of the radical appears to be catalyzed by the microsomal components. In the absence of AA, the intensity of the ESR signal decreased to 16 ± 15% of the complete reaction mixture, suggesting that the radical is derived from AA. For the complete reaction mixture with boiled microsomes, the intensity of the ESR signal decreased to 49 ± 4% of the complete reaction mixture. The intensity of the ESR signal of the complete reaction mixture with indomethacin decreased to 74 ± 20% of the complete reaction mixture, suggesting that cyclooxygenese partly participates in the reaction. A peak was detected on the elution profile of HPLC-ESR analysis of the complete reaction mixture. To determine the structure of the peak, an HPLC-ESR-MS analysis was performed. The HPLC-ESR-MS analysis of the peak showed two prominent ions, m/z 266 and m/z 179, suggesting that the peak is a 4-POBN/pentyl radical adduct. An HPLC-ESR analysis of the authentic 4-POBN/pentyl radical adduct comfirmed the identification.

In Vitro Analysis of Hrd1p-mediated Retrotranslocation of Its Multispanning Membrane Substrate 3-Hydroxy-3-methylglutaryl (HMG)-CoA Reductase

Endoplasmic reticulum (ER)-associated degradation (ERAD) is responsible for the ubiquitin-mediated destruction of both misfolded and normal ER-resident proteins. ERAD substrates must be moved from the ER to the cytoplasm for ubiquitination and proteasomal destruction by a process called retrotranslocation. Many aspects of retrotranslocation are poorly understood, including its generality, the cellular components required, the energetics, and the mechanism of transfer through the ER membrane. To address these questions, we have developed an in vitro assay, using the 8-transmembrane span ER-resident Hmg2p isozyme of HMG-CoA reductase fused to GFP, which undergoes regulated ERAD mediated by the Hrd1p ubiquitin ligase. We have now directly demonstrated in vitro retrotranslocation of full-length, ubiquitinated Hmg2p-GFP to the aqueous phase. Hrd1p was rate-limiting for Hmg2p-GFP retrotranslocation, which required ATP, the AAA-ATPase Cdc48p, and its receptor Ubx2p. In addition, the adaptors Dsk2p and Rad23p, normally implicated in later parts of the pathway, were required. Hmg2p-GFP retrotranslocation did not depend on any of the proposed ER channel candidates. To examine the role of the Hrd1p transmembrane domain as a retrotranslocon, we devised a self-ubiquitinating polytopic substrate (Hmg1-Hrd1p) that undergoes ERAD in the absence of Hrd1p. In vitro retrotranslocation of full-length Hmg1-Hrd1p occurred in the absence of the Hrd1p transmembrane domain, indicating that it did not serve a required channel function. These studies directly demonstrate polytopic membrane protein retrotranslocation during ERAD and delineate avenues for mechanistic understanding of this general process.

Synthesis and characterization of polyoxometalates loaded starch nanocomplex and its antitumoral activity

According to a water-in-oil emulsion technique using POCl 3 as a cross-linking agent, starch nanoparticles have been prepared. Using this starch nanoparticles, loaded polyoxometalates (POMs) α-K 8H 6 [Si 2W 18Ti 6O 77] (Si 2W 18Ti 6) have been prepared and structurally characterized by elemental analyses, IR spectroscopy and NMR spectroscopy. The result shows that the Si 2W 18Ti 6 molecule retains its parent structure after encapsulated by starch nanoparticles and Si 2W 18Ti 6 incorporates into starch matrix to form the nanoparticles. This new complex exhibits higher stability, antitumoral activity and can extend the survival days of the mice with tumor owing to the encapsulation by starch nanoparticles. The Si 2W 18Ti 6 has tested negative for mutagenicity in the standard Ames assay with TA97, TA98, TA100 and TA102, indicating that Si 2W 18Ti 6 has no mutagenic effect in the microsome suspension assay.

Effect of Low pH on the Susceptibility of Isolated Cod (Gadus morhua) Microsomes to Lipid Oxidation

During the extraction of muscle to produce protein isolates by acid or alkali solubilization, membranes are exposed to abnormally low or high pH. Low but not high pH treatment induces rapid oxidation of membrane phospholipids in the presence of hemoglobin. The goal of this research work was to study the oxidative stability of microsomes under the conditions met during acid solubilization. Isolated microsomes from cod muscle were used as a model system. At pH 5.3 or lower, 99% of isolated cod membranes sedimented at low centrifugation speeds. Isolated membranes that were exposed to pH 3.0 were less susceptible to hemoglobin-mediated lipid oxidation. Cod hemoglobin exposed to pH 3 was rendered less pro-oxidative than the untreated cod hemoglobin. However, when microsomes and hemoglobin were together exposed to low pH, oxidation was promoted. Citric acid and calcium chloride, as well as press juice isolated from cod muscle, were able to inhibit lipid oxidation of microsomal suspensions.

Topology and Membrane Association of Lecithin: Retinol Acyltransferase

Fatty acid retinyl esters are the storage form of vitamin A (all-trans-retinol) and serve as metabolic intermediates in the formation of the visual chromophore 11-cis-retinal. Lecithin:retinol acyltransferase (LRAT), the main enzyme responsible for retinyl ester formation, acts by transferring an acyl group from the sn-1 position of phosphatidylcholine to retinol. To define the membrane association and localization of LRAT, we produced an LRAT-specific monoclonal antibody, which we used to study enzyme partition under different experimental conditions. Furthermore, we examined the membrane topology of LRAT through an N-linked glycosylation scanning approach and protease protection assays. We show that LRAT is localized to the membrane of the endoplasmic reticulum (ER) and assumes a single membrane-spanning topology with an N-terminal cytoplasmic/C-terminal luminal orientation. In eukaryotic cells, the C-terminal transmembrane domain is essential for the activity and ER membrane targeting of LRAT. In contrast, the N-terminal hydrophobic region is not required for ER membrane targeting or enzymatic activity, and its amino acid sequence is not conserved in other species examined. We present experimental evidence of the topology and subcellular localization of LRAT, a critical enzyme in vitamin A metabolism.

Danazol inhibits aromatase activity of endometriosis-derived stromal cells by a competitive mechanism

To evaluate the inhibitory effect of danazol on estrogen (E) production in endometriosis. Prospective randomized study. Academic research unit of the department of obstetrics and gynecology in a university hospital. Thirteen patients with endometriosis. Danazol was added to the culture of endometriosis-derived stromal cells or suspensions of microsomes prepared from chocolate cysts. The aromatase activities as well as mRNA and protein levels of aromatase in endometriosis-derived stromal cells or microsomes of endometriosis were examined. Danazol treatment with a concentration greater than 10(-6) M significantly suppressed aromatase activity of endometriosis-derived stromal cells under basal and prostaglandin E(2) (PGE(2))-stimulated conditions. Danazol (10(-5) M) did not affect mRNA and protein levels of aromatase. Danazol competitively inhibited aromatase activity (by 1.7 x 10(-6) M of calculated Ki and 2.9 x 10(-5) M of Ki') of endometriosis microsomes. Danazol competitively inhibited aromatase activity in endometriosis-derived stromal cells without affecting either the mRNA or protein levels of aromatase. These results indicate the efficacy of local application of danazol to endometriotic lesions.

Validation of a HPLC method for the determination of p-nitrophenol hydroxylase activity in rat hepatic microsomes

We report a HPLC-UV method for determination of p-nitrophenol (PNP) hydroxylation to 4-nitrocatechol (4NC) as a marker for CYP2E1 activity in rat hepatic microsomes. Proteins were precipitated by addition of 50 microL phosphoric acid (50%, v/v in water) to 500 microL microsomal suspensions. Following vortex mixing and centrifugation the supernatant (20 microL) was injected onto a Supelcosil C(18) column (150 mm x 4.6 mm, 5 microm), and mobile phase (22% acetonitrile, 0.1% trifluoroacetic acetic acid, 0.5% triethylamine) delivered at 1.0 mL/min produced resolved peaks for internal standard, 4NC, and PNP in < 11 min. Calibration curves were linear (r(2) = 0.999) from 0.1 to 40 microM with intra- and inter-day precision < 12% and accuracy >90%. The method's improved sensitivity (LOQ = 0.1 microM) and minimal sample processing allowed rapid monitoring of PNP hydroxylase activity in fetal, neonatal, juvenile, and adult rat livers.

Comparison of gene expression in cell culture to that in the intact animal: relevance to drugs and environmental toxicants. Focus on “Development of a transactivator in hepatoma cells that allows expression of phase I, phase II, and chemical defense genes”

to study the effects of drugs and other environmental toxicants on the liver, many believe that the use of hepatocyte-derived cultures offers advantages over studies in the liver of intact animals. Cell cultures can be manipulated more easily, and they are far less expensive to maintain than a

Preparation of trout liver microsomes for iron speciation in P-450 enzymes by AE?FPLC with ICP?(ORS)MS detection

Cytochromes P-450 are members of a superfamily of hemoproteins involved in the oxidative metabolism of various physiological and xenobiotic compounds in eukaryotes and prokaryotes. The multiplicity of this group of enzymes has been widely studied by chromatographic techniques, mainly high-performance liquid chromatography (HPLC). Because these enzymes are membrane-bound proteins, sample preparation for chromatographic separation of P-450 enzymes requires a solubilization step. The sample-preparation procedures are critical, because detergents affect not only the efficiency of protein solubilization but also their further chromatographic resolution. Trout liver microsomes have been taken here as a model sample to investigate iron speciation in cytochrome P-450. Trouts were treated intraperitoneally with beta-naphthoflavone, a potent inducer of some P-450 enzymes, and a microsomal suspension containing 7.4+/-0.1 nmol mL(-1) P-450 enzymes was obtained by ultracentrifugation. Lubrol PX was selected as detergent for solubilization, resulting in about 90% solubilization recovery. The solubilized cytochromes P-450 were further separated by AE-FPLC, with UV detection, or coupled to ICP-MS with an octapole reaction system, ICP-(ORS)MS (monitoring Fe signals at masses 54, 56, and 57). A sampling procedure and chromatographic conditions are developed and were successfully applied to iron speciation in trout liver P-450 enzymes. ICP-(ORS)MS detection of P-450 enzymes is Fe-specific and so will give accurate information on the prosthetic group of the protein, which can constitute an advantageous alternative to classical methods for detection of these hemoproteins.

Identification of 2,3-diaminophenazine and of o-benzoquinone dioxime as the major in vitro metabolites of benzofuroxan

The results of an in vitro study of the metabolism of benzofuroxan using either cytosolic or microsomal fractions obtained from rat liver are reported.Benzofuroxan was incubated with an appropriate volume of cytosol or microsomal suspension; control incubations were performed without the β-nicotinamide adenine dinucleotide phosphate-generating system or, alternatively, by using the subcellular fractions inactivated by heating. Incubation mixtures were analysed by high-performance liquid chromatography. Two principal metabolites (M1, M2) were identified in the cytosolic fraction only. The dependence of M2 formation on thiol cofactors, incubation time and protein concentration was examined.The two metabolites were isolated and characterized by their 1H-, 13C-nuclear magnetic resonance, infrared and mass spectra. The structures of o-benzoquinonedioxime (2) and 2,3-diaminopleuozuc (3), were arranged to M1 and M2 respectively. The proposed structures were confirmed by the identity of the metabolites with authentic samples obtained by synthesis. X-ray analysis showed that the dioxime metabolite had an amphy configuration.A metabolic scheme for the formation of the two products is proposed.