-
Journal of Chromatography. B,... Sep 2014This paper describes an extension and performance characterization of a quantitative confirmatory multi-residue liquid chromatography-tandem mass spectrometric method...
Performance characterization of a quantitative liquid chromatography-tandem mass spectrometric method for 12 macrolide and lincosamide antibiotics in salmon, shrimp and tilapia.
This paper describes an extension and performance characterization of a quantitative confirmatory multi-residue liquid chromatography-tandem mass spectrometric method for residues of macrolide and lincosamide antibiotics, originally validated for application to bovine kidney tissues, to tissues of salmon, shrimp and tilapia. The 12 analytes include clindamycin, erythromycin A, gamithromycin, josamycin, lincomycin, neospiramycin 1, oleandomycin, pirlimycin, spiramycin 1, tildipirosin, tilmicosin and tylosin A. The limit of detection was 0.5 μg/kg. Within-laboratory precision evaluated over the analytical range of 5.0-50.0 μg/kg ranged from 4 to 17%. The accuracy of the method ranged from 80 to 112%. Recoveries ranged from 47 to 99% with all but one recovery above 60%. This is the first report of a quantitative confirmatory method for gamithromycin, pirlimycin and tildipirosin in fish and shrimp.
Topics: Animals; Anti-Bacterial Agents; Chromatography, Liquid; Drug Residues; Limit of Detection; Lincosamides; Linear Models; Macrolides; Penaeidae; Reproducibility of Results; Salmon; Seafood; Tandem Mass Spectrometry; Tilapia
PubMed: 25125397
DOI: 10.1016/j.jchromb.2014.07.031 -
Journal of Medical Microbiology Nov 2014In this study, the species diversity of staphylococci with inducible resistance to macrolides, lincosamides and streptogramin B (MLSB) isolated from clinical samples,...
In this study, the species diversity of staphylococci with inducible resistance to macrolides, lincosamides and streptogramin B (MLSB) isolated from clinical samples, sewage and river water was investigated. Inducible clindamycin resistance was tested using a D-test and macrodilution assays. Inducible cross-resistance (iMLSB phenotype) was examined by PCR of erm gene classes A, B, C, F, G, Q, T and 43. Although ermC was the most frequently detected resistance gene in iMLSB phenotypes of environmental staphylococci (61.2%), resistance genes encoding iMLSB were more diverse than in staphylococci from hospital samples. In 22.4% of iMLSB staphylococci from aquatic environments, none of the eight tested erm genes was found. Those isolates and erm43-expressing Staphylococcus lentus displayed low erythromycin MICs (3-16 µg ml(-1)) compared with ermC-positive environmental staphylococci (≥256 µg ml(-1)). In contrast to clinical isolates with clearly defined resistance behaviour, resistance patterns against MLSB and MICs for clindamycin of environmental isolates were more diverse. Although the abundance of iMLSB staphylococci in the aquatic environment was lower than in staphylococci from hospital samples, the diversity of resistance genes encoding this phenotype seemed to be higher. Oleandomycin is the best marker to correlate iMLSB phenotype and the respective erm gene. The phenotypical behaviour of environmental isolates may differ from the resistance pattern of clinical iMLSB staphylococci expressing ermA or ermC, and this should be considered for successful treatment of infections.
Topics: Anti-Bacterial Agents; Clindamycin; Drug Resistance, Bacterial; Humans; Microbial Sensitivity Tests; Species Specificity; Staphylococcal Infections; Staphylococcus; Water Microbiology
PubMed: 25106860
DOI: 10.1099/jmm.0.077081-0 -
International Journal of Toxicology Jul 2014The activities of different xenobiotic-metabolizing enzymes in liver subcellular fractions from Wistar rats exposed to a glyphosate (GLP)-based herbicide (Roundup full... (Comparative Study)
Comparative Study
The activities of different xenobiotic-metabolizing enzymes in liver subcellular fractions from Wistar rats exposed to a glyphosate (GLP)-based herbicide (Roundup full II) were evaluated in this work. Exposure to the herbicide triggered protective mechanisms against oxidative stress (increased glutathione peroxidase activity and total glutathione levels). Liver microsomes from both male and female rats exposed to the herbicide had lower (45%-54%, P < 0.01) hepatic cytochrome P450 (CYP) levels compared to their respective control animals. In female rats, the hepatic 7-ethoxycoumarin O-deethylase (a general CYP-dependent enzyme activity) was 57% higher (P < 0.05) in herbicide-exposed compared to control animals. Conversely, this enzyme activity was 58% lower (P < 0.05) in male rats receiving the herbicide. Lower (P < 0.05) 7-ethoxyresorufin O-deethlyase (EROD, CYP1A1/2 dependent) and oleandomycin triacetate (TAO) N-demethylase (CYP3A dependent) enzyme activities were observed in liver microsomes from exposed male rats. Conversely, in females receiving the herbicide, EROD increased (123%-168%, P < 0.05), whereas TAO N-demethylase did not change. A higher (158%-179%, P < 0.01) benzyloxyresorufin O-debenzylase (a CYP2B-dependent enzyme activity) activity was only observed in herbicide-exposed female rats. In herbicide-exposed rats, the hepatic S-oxidation of methimazole (flavin monooxygenase dependent) was 49% to 62% lower (P < 0.001), whereas the carbonyl reduction of menadione (a cytosolic carbonyl reductase-dependent activity) was higher (P < 0.05). Exposure to the herbicide had no effects on enzymatic activities dependent on carboxylesterases, glutathione transferases, and uridinediphospho-glucuronosyltransferases. This research demonstrated certain biochemical modifications after exposure to a GLP-based herbicide. Such modifications may affect the metabolic fate of different endobiotic and xenobiotic substances. The pharmacotoxicological significance of these findings remains to be clarified.
Topics: 7-Alkoxycoumarin O-Dealkylase; Animals; Carbonyl Reductase (NADPH); Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1A2; Cytochrome P-450 CYP2B1; Cytochrome P-450 CYP3A; Dose-Response Relationship, Drug; Female; Glycine; Herbicides; Liver; Male; Microsomes, Liver; Organophosphate Poisoning; Oxidative Stress; Oxygenases; Random Allocation; Rats, Wistar; Sex Characteristics; Water Pollutants, Chemical; Xenobiotics; Glyphosate
PubMed: 24985121
DOI: 10.1177/1091581814540481 -
International Journal of Systematic and... Sep 2014A novel Gram-staining-positive, aerobic bacterium, designed DCY80(T), was isolated from soil of a ginseng field in the Republic of Korea. 16S rRNA gene sequence analysis...
A novel Gram-staining-positive, aerobic bacterium, designed DCY80(T), was isolated from soil of a ginseng field in the Republic of Korea. 16S rRNA gene sequence analysis revealed that strain DCY80(T) belonged to the genus Brachybacterium (95.8-98.2 % similarity) and was most closely related to Brachybacterium faecium DSM 4810(T) (98.2 %). Colonies were circular, entire, low-convex, opaque and 0.5-1.0 mm in diameter after growth for 2 days on TSA at 30 °C. Growth occurred at 4-34 °C (optimum, 25 °C), at pH 5.0-10.0 (optimum, pH 6.5-7.0) and in the presence of 0-7.0 % NaCl. Strain DCY80(T) produced siderophores and was sensitive to penicillin G, erythromycin, cefazolin, oleandomycin, ceftazidime, vancomycin, tetracycline, novobiocin, carbamicillin, rifampicin and neomycin. The DNA G+C content was 71.0 mol%. Levels of DNA-DNA relatedness between strain DCY80(T) and B. faecium DSM 4810(T), B. paraconglomeratum KCTC 9916(T), B. saurashtrense DSM 23186(T) and B. conglomeratum KCTC 9915(T) were 46.9±0.5, 28.9±0.6, 20.4±0.9 and 17.3±0.4 %, respectively. The cell-wall peptidoglycan of strain DCY80(T) contained meso-diaminopimelic acid as the diagnostic diamino acid. The menaquinones were MK-7 (85.8 %) and MK-8 (14.2 %). The major cellular fatty acids were anteiso-C15 : 0 (69.1 %) and anteiso-C17 : 0 (12.2 %). Phosphatidylglycerol, diphosphatidylglycerol, an unidentified glycolipid, two unidentified phospholipids and five unidentified polar lipids were found. On the basis of our phenotypic and genotypic analyses, strain DCY80(T) represents a novel species of the genus Brachybacterium, for which the name Brachybacterium ginsengisoli sp. nov. is proposed (type strain DCY80(T) = KCTC 29226(T) = JCM 19356(T)).
Topics: Actinomycetales; Base Composition; Cell Wall; DNA, Bacterial; Diaminopimelic Acid; Fatty Acids; Molecular Sequence Data; Nucleic Acid Hybridization; Panax; Peptidoglycan; Phospholipids; Phylogeny; RNA, Ribosomal, 16S; Republic of Korea; Sequence Analysis, DNA; Soil Microbiology; Vitamin K 2
PubMed: 24944333
DOI: 10.1099/ijs.0.058388-0 -
Biochemical Pharmacology Aug 2013Drug-drug interactions (DDIs) may cause serious drug toxicity and delay development of candidate drugs. Screening using human liver microsomes and hepatocytes can help...
Drug-drug interactions (DDIs) may cause serious drug toxicity and delay development of candidate drugs. Screening using human liver microsomes and hepatocytes can help predict DDIs but do not always provide the degree of certainty required for confident progression of a candidate drug. Thus a suitable in vivo test system could be of great value. Here a Cyp2c knockout (KO) mouse was investigated for studying DDIs using midazolam (MDZ) a standard human CYP3A4 substrate and troleandomycin (TAO) a potent human CYP3A4 inhibitor. Pharmacokinetics (PK) and biotransformation of MDZ were investigated following dosing to Cyp2c KO and wild type mice before and after TAO treatment. The noteworthy differences in the metabolism of MDZ in Cyp2c KO compared to wild type mice confirms the important role that Cyp2c enzymes play in the murine metabolism of MDZ in vivo. The impact of Cyp3a inhibition produced a further increase in circulating MDZ concentrations in all individuals from both strains of mice though the impact of the elimination of the Cyp2c pathway in the KO mice on the AUC was less than perhaps expected. We have shown that TAO produces an increase in the MDZ concentration and a reduction in the 1'hydroxymidazolam/midazolam formation ratio but the expected difference in the magnitude of this effect between the wild type and the Cyp2c KO mice was not seen. The magnitude of the TAO effect was also smaller than is reported in humans. Hence further work is required before this animal model could be used to predict clinical interactions.
Topics: Animals; Bile; Biotransformation; Cytochrome P-450 CYP3A; Cytochrome P-450 CYP3A Inhibitors; Cytochrome P-450 Enzyme System; Drug Interactions; Mice; Mice, Knockout; Midazolam; Troleandomycin
PubMed: 23732297
DOI: 10.1016/j.bcp.2013.05.015 -
Drug Metabolism and Pharmacokinetics 2013Effects of green tea extract (GTE) on the activity of cytochrome P450 (CYP) enzymes and pharmacokinetics of simvastatin (SIM) were investigated in rats. Inhibitory...
Effects of green tea extract (GTE) on the activity of cytochrome P450 (CYP) enzymes and pharmacokinetics of simvastatin (SIM) were investigated in rats. Inhibitory effects of GTE on CYP3A activity were investigated in rat hepatic microsomes (RHM) using midazolam (MDZ) 1'-hydroxylation as a probe reaction. SD female rats received a single oral dose of GTE (400 mg/kg) or troleandomycin (TAO, a CYP3A selective inhibitor, 500 mg/kg), followed 30 min later by SIM (20 mg/kg). Plasma concentrations of SIM and its active metabolite, simvastatin acid, were determined up to 6 h after the SIM administration using LC/MS/MS. In RHM, GTE inhibited MDZ 1'-hydroxylation with IC₅₀ and K(i)(app) values of 12.5 and 18.8 µg/mL, respectively, in a noncompetitive manner. Area under plasma concentration-time curves for SIM in the GTE and TAO groups were increased by 3.4- and 10.2-fold, respectively, compared with the control. The maximum concentrations of SIM were higher in the GTE (3.3-fold) and TAO (9.5-fold) groups. GTE alters the pharmacokinetics of SIM, probably by inhibiting intestinal CYP3A.
Topics: Animals; Camellia sinensis; Cytochrome P-450 CYP3A; Cytochrome P-450 CYP3A Inhibitors; Female; Microsomes, Liver; Midazolam; Plant Extracts; Rats; Simvastatin; Troleandomycin
PubMed: 23698259
DOI: 10.2133/dmpk.dmpk-13-nt-006 -
Neuro Endocrinology Letters 2012Of several enzymes metabolizing xenobiotics, cytochrome P450 (CYP) and peroxidase enzymes seem to be most important. One of the major challenges in studies investigating... (Comparative Study)
Comparative Study
OBJECTIVES
Of several enzymes metabolizing xenobiotics, cytochrome P450 (CYP) and peroxidase enzymes seem to be most important. One of the major challenges in studies investigating metabolism of xenobiotics is to resolve which of these two groups of enzymes is predominant to metabolize individual xenobiotic compounds. Utilization of selective inhibitors of CYP and peroxidase enzymes might be a useful tool to identify the contribution of these enzymes to metabolism of xenobiotics in samples, where both types of enzymes are present. The aim of this study was to investigate specificities of several known CYP inhibitors to these enzymes; whether they inhibit only the CYP enzymes and do not inhibit peroxidases.
METHODS
Since the oxidation of o-anisidine catalyzed by a model peroxidase used, horseradish peroxidase (HRP), is a two-substrate reaction, the inhibition potential of tested chemicals was studied with respect to both peroxidase substrates, o-anisidine and hydrogen peroxide. Initial velocities of o-anisidine oxidation by HRP under various conditions were determined spectrophotometrically.
RESULTS
The CYP inhibitors metyrapone, troleandomycine, disulfiram, sulfaphenazole, quinidine and 1-aminobenzotriazole do not inhibit o-anisidine oxidation catalyzed by HRP. In contrast, ketoconazole, diethyldithiocarbamate, ellipticine, α-naphtoflavone, proadifen SKF525A, piperonylbutoxide, were found to inhibit not only the CYPs, but also the HRP-mediated oxidation of o-anisidine. Interestingly, α-naphtoflavone inhibits oxidation of o-anisidine by HRP with respect to H2O2, but not with respect to o-anisidine. Diethyldithiocarbamate is the most potent peroxidase inhibitor of o-anisidine oxidation with Ki with respect to o-anisidine of 10 μM and Ki with respect to H2O2 of 60 μM, being even the better peroxidase inhibitor than the classical "peroxidase inhibitor" - propyl gallate (Ki with respect to o-anisidine of 60 μM and Ki with respect to H2O2 of 750 μM).
CONCLUSIONS
The results of the present study demonstrate that 1-aminobenzotriazole, a potent inhibitor of various CYP enzymes, seems to be the best candidate suitable for utilization in studies evaluating participation of CYP enzymes in metabolism of xenobiotics in various complex biological materials containing both CYP and peroxidase enzymes. Moreover, precaution to prevent misinterpretation of results is necessary in cases when proadifen SKF525A, piperonylbutoxide, diethyldithiocarbamate, ketoconazole, α-naphtoflavone and ellipticine are used in similar studies (as CYP inhibitors in various complex biological materials containing both CYP and peroxidase enzymes), since these chemicals can except of CYP enzymes inhibit also peroxidase-mediated reactions.
Topics: Benzoflavones; Cytochrome P-450 Enzyme Inhibitors; Disulfiram; Ditiocarb; Ellipticines; Enzyme Activation; Enzyme Inhibitors; Horseradish Peroxidase; Humans; Ketoconazole; Metyrapone; Piperonyl Butoxide; Proadifen; Quinidine; Structure-Activity Relationship; Substrate Specificity; Sulfaphenazole; Triazoles; Troleandomycin
PubMed: 23353841
DOI: No ID Found -
Xenobiotica; the Fate of Foreign... Jun 20131. This work investigated the drug interaction potential of GSK1292263, a novel GPR119 agonist, with the HMG-coA reductase inhibitors simvastatin and rosuvastatin. 2. In... (Clinical Trial)
Clinical Trial
1. This work investigated the drug interaction potential of GSK1292263, a novel GPR119 agonist, with the HMG-coA reductase inhibitors simvastatin and rosuvastatin. 2. In vitro experiments assessed the inhibition of transporters and CYP enzymes by GSK1292263, and a clinical drug interaction study investigated the effect of GSK1292263 (300 mg BID) on the pharmacokinetic profile of simvastatin (40 mg single dose) and rosuvastatin (10 mg single dose). 3. In vitro, GSK1292263 demonstrated little/weak inhibition (IC50 values >30 μM) towards CYPs (CYP1A2, 2C9, 2C19, 2D6, 3A4), Pgp, OATP1B3, or OCT2. However, GSK1292263 inhibited BCRP and OATP1B1, which are transporters involved in statin disposition. 4. In the clinical study, small increases in the AUC(0-inf) of simvastatin [mean ratio (90% CI) of 1.34 (1.22, 1.48)] and rosuvastatin [mean ratio (90% CI) of 1.39 (1.30, 1.49)] were observed when co-administered with GSK1292263, which is consistent with an inhibitory effect on intestinal BCRP and CYP3A4. In contrast, GSK1292263 did not inhibit OATP1B1 based on the lack of changes in simvastatin acid exposure [mean AUC(0-inf) ratio (90% CI) of 1.05 (0.91, 1.21)]. 5. GSK1292263 has a weak drug interaction with simvastatin and rosuvastain. This study provides a mechanistic understanding of the in vivo inhibition of transporters and enzymes by GSK1292263.
Topics: Adolescent; Adult; Aged; Animals; Atorvastatin; Cytochrome P-450 CYP3A; Cytochrome P-450 CYP3A Inhibitors; Demography; Dogs; Dose-Response Relationship, Drug; Drug Interactions; Female; Fluorobenzenes; Heptanoic Acids; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Madin Darby Canine Kidney Cells; Male; Mesylates; Middle Aged; Oxadiazoles; Piperidines; Pyrimidines; Pyrroles; Reference Standards; Rosuvastatin Calcium; Simvastatin; Sulfonamides; Troleandomycin; Young Adult
PubMed: 23256625
DOI: 10.3109/00498254.2012.739719 -
Molecular Microbiology Dec 2012MacB is a founding member of the Macrolide Exporter family of transporters belonging to the ATP-Binding Cassette superfamily. These proteins are broadly represented in...
MacB is a founding member of the Macrolide Exporter family of transporters belonging to the ATP-Binding Cassette superfamily. These proteins are broadly represented in genomes of both Gram-positive and Gram-negative bacteria and are implicated in virulence and protection against antibiotics and peptide toxins. MacB transporter functions together with MacA, a periplasmic membrane fusion protein, which stimulates MacB ATPase. In Gram-negative bacteria, MacA is believed to couple ATP hydrolysis to transport of substrates across the outer membrane through a TolC-like channel. In this study, we report a real-time analysis of concurrent ATP hydrolysis and assembly of MacAB-TolC complex. MacB binds nucleotides with a low millimolar affinity and fast on- and off-rates. In contrast, MacA-MacB complex is formed with a nanomolar affinity, which further increases in the presence of ATP. Our results strongly suggest that association between MacA and MacB is stimulated by ATP binding to MacB but remains unchanged during ATP hydrolysis cycle. We also found that the large periplasmic loop of MacB plays the major role in coupling reactions separated in two different membranes. This loop is required for MacA-dependent stimulation of MacB ATPase and at the same time, contributes to recruitment of TolC into a trans-envelope complex.
Topics: ATP-Binding Cassette Transporters; Adenosine Triphosphatases; Adenosine Triphosphate; Bacterial Outer Membrane Proteins; Erythromycin; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Hydrolysis; Kinetics; Macrolides; Membrane Transport Proteins; Microbial Sensitivity Tests; Mutation; Oleandomycin; Protein Binding; Protein Conformation
PubMed: 23057817
DOI: 10.1111/mmi.12046 -
Journal of AOAC International 2012An LC/MS/MS-based multiresidue quantitative method was developed for the macrolides erythromycin A, neospiramycin I, oleandomycin, spiramycin I, tilmicosin, and tylosin...
An LC/MS/MS-based multiresidue quantitative method was developed for the macrolides erythromycin A, neospiramycin I, oleandomycin, spiramycin I, tilmicosin, and tylosin A in porcine kidney tissues. The Canadian Food Inspection Agency (CFIA) had as part of its analytical scope an LC/UV method for quantification of residues of two macrolide antibiotics, tilmicosin and tylosin A, in the kidney, liver, and muscle of cattle, swine, and poultry. The method could not reliably detect concentrations below 10 microg/kg. To increase the scope of the CFIA's analytical capabilities, a sensitive multiresidue quantitative method for macrolide residues in food animal tissues was required. Porcine kidney samples were extracted with acetonitrile and alkaline buffer and cleaned-up using silica-based C18 SPE cartridges. Sample extracts were analyzed using LC/MS/MS with positive electrospray ionization. Fitness for purpose was verified in a single-laboratory validation study using a second analyst. The working analytical range was 5 to 50 microg/kg. LOD and LOQ were 0.5 to 0.6 microg/kg and 1.5 to 3.0 microg/kg, respectively. Limits of identification were 0.5 to 2.0 microg/kg. Relative intermediate precisions were 8 to 17%. Average absolute recoveries were 68 to 76%.
Topics: Animals; Anti-Bacterial Agents; Chromatography, Liquid; Kidney; Macrolides; Molecular Structure; Reproducibility of Results; Sensitivity and Specificity; Swine; Tandem Mass Spectrometry
PubMed: 22649946
DOI: 10.5740/jaoacint.11-139