Search results for "Enzyme Induction"
showing 10 items of 103 documents
Strategies to In Vitro Assessment of Major Human CYP Enzyme Activities by Using Liquid Chromatography Tandem Mass Spectrometry
2008
At the early stage of drug discovery, thousands of new chemical entities (NCEs) may be screened before a single candidate can be identified for development. Determining the role of CYP enzymes in the metabolism of a compound and evaluating the effect of NCEs on human CYP activities are key issues in pharmaceutical development as they may explain inter-subject variability, drug-drug interactions, non-linear pharmacokinetics and toxic effects. Reliable methods for determining enzyme activities are needed to characterize an individual CYP enzyme and to obtain a tool for the evaluation of its role in drug metabolism in humans. Different liquid chromatography tandem mass spectrometry methodologi…
Cryopreserved primary hepatocytes as a constantly available in vitro model for the evaluation of human and animal drug metabolism and enzyme inductio…
2000
The use of primary hepatocytes is now well established for both studies of drug metabolism and enzyme induction. Cryopreservation of primary hepatocytes decreases the need for fresh liver tissue. This is especially important for research with human hepatocytes because availability of human liver tissue is limited. In this review, we summarize our research on optimization and validation of cryopreservation techniques. The critical elements for successful cryopreservation of hepatocytes are (1) the freezing protocol, (2) the concentration of the cryoprotectant [10% dimethyl-sulfoxide (DMSO)], (3) slow addition and removal of DMSO, (4) carbogen equilibration during isolation of hepatocytes and…
Heme oxygenase-1 inhibits apoptosis in Caco-2 cells via activation of Akt pathway
2005
Heme oxygenase-1 can play a protective role against cellular stress. In colon cancer cells, these effects would be relevant to oncogenesis and resistance to chemotherapy. The aim of the study was to examine the effects of heme oxygenase-1 induction on cell survival in a human colon cancer cell line, Caco-2. Serum deprivation induced apoptosis, reduced Akt and p38 phosphorylation, and increased p21(Cip/WAF1) levels. Heme oxygenase-1 induction by treatment with cobalt protoporphyrin IX resulted in resistance to apoptosis, activation of Akt, reduction in p21(Cip/WAF1) levels and modification of bcl2/bax ratio towards survival. Indomethacin reduced apoptosis but in contrast to heme oxygenase-1,…
Coordinated induction of drug transporters and phase I and II metabolism in human liver slices
2008
Although regulation of phase I drug metabolism in human liver is relatively well studied, the regulation of phase II enzymes and of drug transporters is incompletely characterized. Therefore, we used human liver slices to investigate the PXR, CAR and AhR-mediated induction of drug transporters and phase I and II metabolic enzymes. Precision-cut human liver slices were incubated for 5 or 24 h with prototypical inducers: phenobarbital (PB) (50 mu M) for CAR, beta-naphthoflavone (BNF) (25 mu M) for AhR, and rifampicin (RIF) (10 mu M) for PXR, and gene expression of the phase I enzymes CYP1A1, 1A2, 3A4, 3A5, 2136, 2A6, the phase II enzymes UGT1A1 and 1A6, and the transporters MRP2, MDR1, BSEP, …
Fen1 is induced p53 dependently and involved in the recovery from UV-light-induced replication inhibition.
2005
Mouse embryonic fibroblasts (MEFs) that lack p53 are hypersensitive to the cytotoxic and genotoxic effect of ultraviolet (UV-C) light. They also display a defect in the recovery from UV-C-induced DNA replication inhibition. An enzyme involved in processing stalled DNA replication forks is flap endonuclease 1 (Fen1). Gene expression profiling of UV-C-irradiated MEFs revealed fen1 to be upregulated, which was confirmed by RT-PCR and Western blot experiments. Increased Fen1 levels upon UV-C exposure are due to transcriptional activation, as revealed by inhibitor studies. Fen1 induction was dose- and time-dependent; it occurred on protein level already 3 h after irradiation. Induction of Fen1 b…
Resveratrol, a chemopreventive agent, disrupts the cell cycle control of human SW480 colorectal tumor cells
2002
Resveratrol is a natural polyphenolic compound produced by a number of plants and found in high amount in peanuts, seeds, grapes or berries as source of human nutrition. Epidemiological studies strongly suggest that resveratrol may act as a cancer chemopreventive compound. The mechanism by which resveratrol inhibits cell proliferation was studied in human colorectal tumor SW480 cell line. The results show that resveratrol strongly inhibits cell proliferation at the micromolar range in a time- and dose-dependent manner. Resveratrol appears to block the cell cycle at the transition --> G2/M since inhibition of [(3)H]-thymidine incorporation is not observed, while there is an increase of the c…
Replication of HSV-1 in murine peritoneal macrophages: comparison of various virus strains with different properties.
1984
The in vitro replication of eleven different strains of herpes simplex virus type 1 was studied in resident or thioglycollate-stimulated mouse macrophages. The strains of herpes simplex virus differed in the type of cytopathic effect, induction capacity for herpes simplex virus coded thymidine kinase and pathogenicity in the mouse. Herpes simplex virus replicated better in thioglycollate-stimulated macrophages than in resident macrophages. In vitro ageing of macrophages increased their replicative potency. Herpes simplex virus replicated better in macrophages from homozygous bg/bg C57/BL6J mice than in macrophages from their heterozygous littermates. Separation of macrophages on discontinuo…
Quantitative RT-PCR measurement of human cytochrome P-450s: application to drug induction studies.
2000
A quantitative RT-PCR assay has been developed that is able to measure the mRNA content of the major human CYPs (1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5). The technique is highly specific, reproducible, rapid, and sensitive enough to quantitate low and high abundant mRNAs. The PCR primers were selected to specifically match each CYP mRNA, to have a very close annealing temperature, and to render PCR products of similar sizes. The PCR conditions were designed to allow the simultaneous measurement of the various human liver CYPs in a single run. To achieve precise and reproducible quantitation of each cytochrome mRNA, a external standard (luciferase mRNA) is added to the probes …
Differential sensitivity of rat hepatocyte CYP isoforms to self-generated nitric oxide.
2001
AbstractEarly loss of P450 in rat hepatocyte cultures appears directly related to nitric oxide (NO) overproduction. This study investigates the influence of endogenously generated NO (or NO-derived species) on the relative expression of cytochrome P450 (CYP) isoforms in rat hepatocytes. Our results support the view that loss of P450 holoenzyme in culture is the ultimate consequence of a NO driven process, activated during the common hepatocyte isolation procedure, that leads to an accelerated and selective degradation of specific CYP apoproteins. Under conditions in which NO and peroxynitrite formation is operative, changes in the level of specific CYP isoforms result in a significant alter…
Semi-automatic quantitative RT-PCR to measure CYP induction by drugs in human hepatocytes
2003
An assay has been developed for the quantitative measurement of CYP mRNA content of the major human isoforms (1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5) in human hepatocytes. The method is based on the conversion of mRNAs into their corresponding cDNAs, followed by PCR amplification using appropriate primers. Making use of appropriate internal and external standards it is possible to estimate changes in CYP mRNA content of hepatocytes. The technique has been standardised to run semi-automatically. This procedure can be used to assess the CYP induction potential of new pharmaceuticals at a pre-clinical stage of development. To this aim, human hepatocytes obtained from functional l…