showing 16 related works from this author
Fluorescence-based assays for screening nine cytochrome P450 (P450) activities in intact cells expressing individual human P450 enzymes.
2004
In this study we describe a battery of fluorescence assays for rapid measurement in intact cells of the activity of nine cytochromes P450 (P450s) involved in drug metabolism. The assays are based on the direct incubation of monolayers of cells expressing individual P450 enzymes with a fluorogenic substrate followed by fluorimetric quantification of the product formed and released into incubation medium. For each individual P450 activity, different fluorescence probes were examined, and the one showing the best properties (highest metabolic rates, lowest background fluorescence) was selected: 3-cyano-7-ethoxycoumarin for CYP1A2 and CYP2C19, coumarin for CYP2A6, 7-ethoxy-4-trifluoromethylcoum…
Evaluation of drug-metabolizing and functional competence of human hepatocytes incubated under hypothermia in different media for clinical infusion.
2008
Hepatocyte transplantation has been proposed as a method to support patients with liver insufficiency. Key factors for clinical cell transplantation to progress is to prevent hepatocyte damage, loss of viability and cell functionality, factors that depend on the nature of the tissue used for isolation to a large extent. The main sources of tissue for hepatocyte isolation are marginal livers that are unsuitable for transplantation, and segments from reduced cadaveric grafts. Hepatocellular transplantation requires infusing human hepatocytes in Suspension over a period of minutes to hours. The beneficial effect of hypothermic preservation of hepatocytes in infusion medium has been reported, b…
High-content imaging technology for the evaluation of drug-induced steatosis using a multiparametric cell-based assay.
2012
In the present study, we developed a cell-based protocol for the identification of drugs able to induce steatosis. The assay measures multiple markers of toxicity in a 96-well plate format using high-content screening (HCS) technology. After treating HepG2 cells with increasing concentrations of the tested compounds, toxicity parameters were analyzed using fluorescent probes: BODIPY493/503 (lipid content), 2',7'-dihydrodichlorofluorescein diacetate (reactive oxygen species [ROS] generation), tetramethyl rhodamine methyl ester (mitochondrial membrane potential), propidium iodide (cell viability), and Hoechst 33342 (nuclei staining). A total of 16 drugs previously reported to induce liver ste…
Virulence factor rtx in Legionella pneumophila, evidence suggesting it is a modular multifunctional protein
2008
Abstract Background The repeats in toxin (Rtx) are an important pathogenicity factor involved in host cells invasion of Legionella pneumophila and other pathogenic bacteria. Its role in escaping the host immune system and cytotoxic activity is well known. Its repeated motives and modularity make Rtx a multifunctional factor in pathogenicity. Results The comparative analysis of rtx gene among 6 strains of L. pneumophila showed modularity in their structures. Among compared genomes, the N-terminal region of the protein presents highly dissimilar repeats with functionally similar domains. On the contrary, the C-terminal region is maintained with a fashionable modular configuration, which gives…
Cytometric analysis for drug-induced steatosis in HepG2 cells
2009
Drugs are capable of inducing hepatic lipid accumulation. When fat accumulates, lipids are primarily stored as triglycerides which results in steatosis and provides substrates for lipid peroxidation. An in vitro multiparametric flow cytometry assay was performed in HepG2 cells by using fluorescent probes to analyze cell viability (propidium iodide, PI), lipid accumulation (BODIPY493/503), mitochondrial membrane potential (tetramethyl rhodamine methyl ester, TMRM) and reactive oxygen species generation (ROS) (2',7'-dihydrochlorofluorescein diacetate, DHCF-DA) as functional markers. All the measurements were restricted to live cells by gating the cells that excluded PI or those that exhibited…
Cryopreservation of rat, dog and human hepatocytes: influence of preculture and cryoprotectants on recovery, cytochrome P450 activities and induction…
2006
Several cryopreservation protocols for hepatocytes have been proposed over the past few years, but their effectiveness varies greatly as a function of the characteristics of the method used. One factor in the success of cryopreservation is the quality of cells before freezing. The results suggest that the cryopreservation of hepatocytes in a medium containing polyvinylpyrrolidone (PVP), in addition to DMSO, constitutes a convenient means of long-term storage of hepatocytes for preparing primary cultures to be used in drug metabolism studies. The combined use of the two cryoprotectants is particularly critical for low-viability cell suspensions. An interesting alternative to increase cell vi…
LC-MS untargeted metabolomic analysis of drug-induced hepatotoxicity in HepG2 cells
2015
Hepatotoxicity is the number one cause for agencies not approving and withdrawing drugs for the market. Drug-induced human hepatotoxicity frequently goes undetected in preclinical safety evaluations using animal models. Human-derived in vitro models represent a common alternative to in vivo tests to detect toxic effects during preclinical testing. Most current in vitro toxicity assays rely on the measurement of nonspecific or low sensitive endpoints, which result in poor concordance with human liver toxicity. Therefore, making more accurate predictions of the potential hepatotoxicity of new drugs remains a challenge. Metabolomics, whose aim is to globally assess all the metabolites present …
Incidence, Clinical Characteristics and Management of Inflammatory Bowel Disease in Spain: Large-Scale Epidemiological Study
2021
(1) Aims: To assess the incidence of inflammatory bowel disease (IBD) in Spain, to describe the main epidemiological and clinical characteristics at diagnosis and the evolution of the disease, and to explore the use of drug treatments. (2) Methods: Prospective, population-based nationwide registry. Adult patients diagnosed with IBD—Crohn’s disease (CD), ulcerative colitis (UC) or IBD unclassified (IBD-U)—during 2017 in Spain were included and were followed-up for 1 year. (3) Results: We identified 3611 incident cases of IBD diagnosed during 2017 in 108 hospitals covering over 22 million inhabitants. The overall incidence (cases/100,000 person-years) was 16 for IBD, 7.5 for CD, 8 for UC, and…
Customised in vitro model to detect human metabolism-dependent idiosyncratic drug-induced liver injury
2017
Drug-induced liver injury (DILI) has a considerable impact on human health and is a major challenge in drug safety assessments. DILI is a frequent cause of liver injury and a leading reason for post-approval drug regulatory actions. Considerable variations in the expression levels of both cytochrome P450 (CYP) and conjugating enzymes have been described in humans, which could be responsible for increased susceptibility to DILI in some individuals. We herein explored the feasibility of the combined use of HepG2 cells co-transduced with multiple adenoviruses that encode drug-metabolising enzymes, and a high-content screening assay to evaluate metabolism-dependent drug toxicity and to identify…
Effects of steatosis on drug-metabolizing capability of primary human hepatocytes.
2007
Abstract The suitability of liver grafts discarded for transplantation because of macrosteatosis for preparing human hepatocyte cultures for in vitro drug metabolism studies has been examined. Lower cell viability and yield of isolation procedure were obtained from fatty livers (>40% steatosis) with respect to normal tissue. Significant reductions in 7-ethoxycoumarin O-deethylation (ECOD) and testosterone oxidations were found in hepatocytes prepared from steatotic livers. The potential impact of lipid accumulation on P450 enzymes was studied in vitro by incubation of cultured hepatocytes with long chain free fatty acids (FFA). Treatment of cells with 0.25–3 mM FFA induced dose-dependent ac…
2004
Background Genetic variability in viral populations is usually estimated by means of polymerase chain reaction (PCR) based methods in which the relative abundance of each amplicon is assumed to be proportional to the frequency of the corresponding template in the initial sample. Although bias in template-to-product ratios has been described before, its relevance in describing viral genetic variability at the intrapatient level has not been fully assessed yet.
Inhibition of human P450 enzymes by natural extracts used in traditional medicine
2008
Different medicinal plants are widely used in Cuba and Mexico to treat several disorders. This paper reports in vitro inhibitory effects on the P450 system of herbal products commonly used by people in Cuba and Mexico in traditional medicine for decades. Experiments were conducted in human liver microsomes. The catalytic activities of CYP1A1/2, 2D6, and 3A4 were measured using specific probe substrates. The Heliopsis longipes extract exhibited a concentration-dependent inhibition of the three enzymes, and similar effects were produced by affinin (an alkamide isolated from the H. longipes extract) and two catalytically reduced alkamides. Mangifera indica L. and Thalassia testudinum extracts,…
Effects of Mangifera indica L. aqueous extract (Vimang) on primary culture of rat hepatocytes.
2007
Abstract Vimang is an aqueous extract from stem bark of Mangifera indica L. (Mango) with pharmacological properties. It is a mixture of polyphenols (as main components), terpenoids, steroids, fatty acids and microelements. In the present work we studied the cytotoxic effects of Vimang on rat hepatocytes, possible interactions of the extract with drug-metabolizing enzymes and its effects on GSH levels and lipid peroxidation. No cytotoxic effects were observed after 24 h exposure to Vimang of up to 1000 μg/mL, while a moderate cytotoxicity was observed after 48 and 72 h of exposure at higher concentrations (500 and 1000 μg/mL). The effect of the extract (50–400 μg/mL) on several P450 isozymes…
Advantageous use of HepaRG cells for the screening and mechanistic study of drug-induced steatosis
2016
Only a few in vitro assays have been proposed to evaluate the steatotic potential of new drugs. The present study examines the utility of HepaRG cells as a cell-based assay system for screening drug-induced liver steatosis. A high-content screening assay was run to evaluate multiple toxicity-related cell parameters in HepaRG cells exposed to 28 compounds, including drugs reported to cause steatosis through different mechanisms and non-steatotic compounds. Lipid content was the most sensitive parameter for all the steatotic drugs, whereas no effects on lipid levels were produced by non-steatotic compounds. Apart from fat accumulation, increased ROS production and altered mitochondrial membra…
A human hepatocellular in vitro model to investigate steatosis
2006
The present study was designed to define an experimental model of hepatocellular steatosis with a fat overaccumulation profile in which the metabolic and cytotoxic/apoptotic effects could be separated. This was accomplished by defining the experimental conditions of lipid exposure that lead to significant intracellular fat accumulation in the absence of overt cytotoxicity, therefore allowing to differentiate between cytotoxic and apoptotic effects. Palmitic (C16:0) and oleic (Cl 8: 1) acids are the most abundant fatty acids (FFAs) in liver triglycerides in both normal subjects and patients with nonalcoholic fatty liver disease (NAFLD). Therefore, human hepatocytes and HepG2 cells were incub…
Neonatal Livers: A Source for the Isolation of Good-Performing Hepatocytes for Cell Transplantation
2014
Hepatocyte transplantation is an alternative therapy to orthotopic liver transplantation for the treatment of liver diseases. However, the supply of hepatocytes is limited given the shortage of organs available to isolate good-functioning quality cells. Neonatal livers may be a potential source alternative to adult livers to obtain good-performing hepatic cells for hepatocyte transplantation, which has not yet been explored profoundly. High-yield preparations of viable hepatocytes were isolated from 1- to 23-day-old liver donors, cryopreserved, and banked. Cell integrity and functional quality assessment were performed after thawing. Neonatal hepatocytes showed better postthawing recovery …