Mechanisms of photosensitization by drugs: Involvement of tyrosines in the photomodification of proteins mediated by tiaprofenic acid in vitro

The photosensitizing potential of drugs must be related to their photoreactivity towards the target biomolecules. In this context, a representative photosensitizing drug (tiaprofenic acid) was co-irradiated with a model protein, bovine serum albumin (BSA). This led to a significant degree of protein crosslinking and to the formation of trace amounts of drug-BSA photoadducts. Amino acid analysis of the hydrolysed (HC1) protein showed that His and Tyr undergo a dramatic decrease (approx. 90%) as a consequence of drug-mediated photodynamic processes. When the drug was irradiated in the presence of the pure amino acids, extensive phototransformation of the latter was observed. Other photosensit…

Use of molecular topology in the selection of new cytostatic drugs

Abstract Connectivity indices are the topological descriptors that are able to predict different chemical and biological properties of the organic compounds. Recently, our research group has demonstrated their usefulness in selecting new cytostatic compounds, all of them showing antibacterial activity. In this paper we realize that this ability is considerably increased by using our home-made pharmacological distribution diagrams (PDDs) together with the topological charge indices, so that the efficient selection of new candidates within heterogeneous sets of compounds is possible. This is a straightforward way for the design and/or selection of new active compounds on virtually any type of…

Sequential Hepatogenic Transdifferentiation of Adipose Tissue-Derived Stem Cells: Relevance of Different Extracellular Signaling Molecules, Transcription Factors Involved, and Expression of New Key Marker Genes

Adipose tissue contains a mesenchymal stein cell (MSC) population Known as adipose-derived stein cells (ASCs) capable of differentiating into different cell types. Our aim was to induce hepatic transdifferentiation of ASCs by sequential exposure to several combinations of cytokines, growth factors, and hormones. The most efficient hepatogenic protocol includes fibroblastic growth factors (FGF) 2 and 4 and epidermal growth factor (EGF) (step 1), hepatocyte growth factor (HGF), FGF2, FGF4, and nicotinamide (Nic) (step 2), and oncostatin M (OSM), dexamethasone (Dex), and insulin-tranferrin-selenium (step 3). This protocol activated transcription factors [GATA6, Hex, CCAAT/enhancer binding prot…

Potential hepatoprotective effects of new Cuban natural products in rat hepatocytes culture.

The protective effects of five Cuban natural products (Mangifera indica L. (MSBE), Erythroxylum minutifolium, Erythroxylum confusum, Thalassia testudinum and Dictyota pinnatifida extracts and mangiferin) on the oxidative damage induced by model toxicants in rat hepatocyte cultures were studied. Cells were pre-incubated with the natural products (5-200 microg/mL) for 24 h. Then hepatotoxins (tert-butyl hydroperoxide, ethanol, carbon tetrachloride and lipopolysaccharide) were individually added and post-incubated for another 24 h. After treatments, cell viability was determined using the MTT assay. Mangiferin and MSBE exhibited the highest cytoprotective potential (EC50 between 50 and 125 mic…

SOC-V-11 New serum miRNA biomarkers to predict liver steatosis by valproic acid in paediatric epileptic patients

Depakine (Valproate, VPA) has been the first line, most-frequently prescribed, anti-epileptic drug in children for the past 50 years. Idiosyncratic hepatotoxicity (iDILI) by VPA has been demonstrated in several case reports, where microvesicular liver steatosis was the most frequent feature. Moreover, more than half of VPA-treated patients could have silent fatty liver as demonstrated by ultrasounds. Extensive experimental studies support that VPA has a high potential to induce steatosis in hepatocytes. However, there is an apparent lack of significant hepatic problems in the Neuropediatric Units, despite transaminitis is not uncommon. One of the reasons could be that iDILI and liver steato…

Functional characterization of hepatocytes for cell transplantation: customized cell preparation for each receptor.

The first indication of hepatocyte transplantation is inborn liver-based metabolic disorders. Among these, urea cycle disorders leading to the impairment to detoxify ammonia and Crigler-Najjar Syndrome type I, a deficiency in the hepatic UDP-glucuronosyltransferase 1A1 present the highest incidence. Metabolically qualified human hepatocytes are required for clinical infusion. We proposed fast and sensitive procedures to determine their suitability for transplantation. For this purpose, viability, attachment efficiency, and metabolic functionality (ureogenic capability, cytochrome P450, and phase II activities) are assayed prior to clinical cell infusion to determine the quality of hepatocyt…

Development of multiparametric cell-based protocol to assess and classify hepatotoxicity potential of drugs

Modulation of P450 enzymes by Cuban natural products rich in polyphenolic compounds in rat hepatocytes.

This paper reports cytotoxic effects and changes in the P450 system after exposing rat hepatocytes to four polyphenol-rich products widely used in Cuban traditional medicine (Mangifera indica L. (MSBE), Thalassia testudinum (Tt), Erythroxylum minutifolium and confusum extracts). Effects of mangiferin, the main polyphenol in MSBE, were also evaluated. Cytotoxicity was assayed by the MTT test after exposure of cells to the products (50-1000 microg/mL) for 24 or 72 h. The results showed that 500 microg/mL MSBE was moderately cytotoxic after 72 h, while mangiferin was not. Marked reductions in cell viability were produced by Erythroxylum extracts at concentrationsor = 200 microg/mL, whereas onl…

Intracellular glutathione in human hepatocytes incubated with S-adenosyl-L-methionine and GSH-depleting drugs

Abstract The present study was undertaken to investigate (a) whether S- adenosyl- L -methionine (SAMe) added to culture medium can increase intracellular glutathione (GSH) levels in human hepatocytes and (b) whether SAMe can prevent the GSH depletion found in human hepatocytes incubated with GSH-depleting drugs (paracetamol, opiates, ethanol). Incubation of hepatocytes with increasing concentrations of SAMe resulted in a dose-dependent elevation of intracellular GSH content, which reached its maximum (35% increase) at 30 μM after 20 h. SAMe, as the only sulfur source in the medium, was efficient in repleting GSH-depleted hepatocytes following treatment with diethyl maleate. Incubation of hu…

A Microassay for Measuring Glycogen in 96-Well-Cultured Cells

Abstract This study describes a rapid, sensitive, and automated spectrophotometric enzymatic microassay that measures the intracellular glycogen of primary cultured hepatocytes and other cultured cells in 96-well plates and can be adapted for other samples that are transferred to these plates. The procedure involves in situ disruption of cells, followed by hydrolysis of glycogen into glucosyl units by fungal glucoamylase (exo-1,4-α- D -glucosidase, EC 3.2.1.3), and glucose determination with the glucose oxidase colorimetric method. The color intensity can be measured in conventional ELISA readers, and the data can be fed to an on-line computer for rapid processing. The advantages of this me…

Transcriptional Regulation of Human CYP3A4 Basal Expression by CCAAT Enhancer-Binding Protein α and Hepatocyte Nuclear Factor-3γ

Cytochrome P450 3A4 (CYP3A4) is involved in the metabolism of more than 50% of currently used therapeutic drugs, yet the mechanisms that control CYP3A4 basal expression in liver are poorly understood. Several putative binding sites for CCAAT/enhancer-binding protein (C/EBP) and hepatic nuclear factor 3 (HNF-3) were found by computer analysis in CYP3A4 promoter. The use of reporter gene assays, electrophoretic mobility shift assays, and site-directed mutagenesis revealed that one proximal and two distal C/EBP alpha binding sites are essential sites for the trans-activation of CYP3A4 promoter. No trans-activation was found in similar reporter gene experiments with a HNF-3 gamma expression vec…

A specific microassay for evaluating hepatic LDH activity in co-cultures of hepatocytes with other cells.

This study describes the development of a simple, rapid and reproducible microassay for determining the intracellular LDH activity of rat hepatocytes present in a co-culture system with other cells. The procedure involves treatment of cellular homogenates with an anti-LDH antiserum that specifically inhibits the LDH activity of rat hepatocytes. The assay is performed in 96-well plates and LDH activity can be measured directly in the same wells using a colorimetric method. The difference in LDH activity values measured before and after antiserum incubation reflects the LDH content of the hepatocytes in the sample. The advantages of this method are the small number of cells required, a reduct…

Effects of steatosis on drug-metabolizing capability of primary human hepatocytes.

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…

Human Hepatocyte Transplantation in Patients with Hepatic Failure Awaiting a Graft

<b><i>Background:</i></b> Hepatocyte transplantation (HT) has the potential to become a promising treatment to temporarily support liver function in patients with liver failure. <b><i>Methods:</i></b> Two patients, who had already received a liver transplant (LT) in the past, with an end-stage liver disease due to recurrent hepatitis C virus cirrhosis, suffering acute-on-chronic liver failure while on the waiting list for an LT, received HT as a bridge to whole-organ retransplantation. After HT and during intensive care unit admission, blood tests and ammonia levels were determined every 12 and 24 h, respectively, before and after each hepatoc…

High Expression of Human CYP2C in Immortalized Human Liver Epithelial Cells

Cell lines stably expressing high levels of single isozymes of human CYP2C genes (CYP2C8, CYP2C9, CYP2C18 and CYP2C19) have been successfully generated by transfecting liver epithelial human cells (THLE) with an appropriate expression vector. To this aim, cDNAs encoding for each CYP2C gene were inserted by blunt-ended cloning into the unique insertion site of the singular expression vector pCMVneo. The recombinant pCMV2C8, pCMV2C9, pCMV2C18 and pCMV2C19 vectors were liposome-mediated transfected into THLE cells. The resulting transgenic cells, designated as T5-2C8, T5-2C9, T5-2C18 and T5-2C19, were cloned and the expression of the ectopic gene, mRNA and protein, was investigated by RT-PCR a…

Determination of major human cytochrome P450s activities in 96-well plates using liquid chromatography tandem mass spectrometry.

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. Evaluation of the effect of NCEs on human CYP450 enzyme activities is a key issue in pharmaceutical development as it may explain inter-subject variability, drug-drug interactions, non-linear pharmacokinetics and toxic effects. A liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method has been developed for the fast and routine analysis of major human CYP450s enzyme activities (CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4) in primary hepatocyte cell cultures. The high sensitivity and selectivity of mass …

Cell Lines: A Tool for In Vitro Drug Metabolism Studies

Primary cultured hepatocytes are a valuable in vitro model for drug metabolism studies. However, their widespread use is greatly hindered by the scarcity of suitable human liver samples. Moreover, the well-known in vitro phenotypic instability of hepatocytes, the irregular availability of fresh human liver for cell harvesting purposes, and the high batch-to-batch functional variability of hepatocyte preparations obtained from different human liver donors, seriously complicate their use in routine testing. To overcome these limitations, different cell line models have been proposed for drug metabolism screening. Human liver-derived cell lines would be ideal models for this purpose given thei…

Cocaine hepatotoxicity: two different toxicity mechanisms for phenobarbital-induced and non-induced rat hepatocytes.

Abstract Hepatocytes isolated from both phenobarbital-induced and control rats were short-term cultured and exposed to cocaine (8–2000 μM) for varying times. Intracellular lactate dehydrogenase activity, free calcium levels ([Ca 2+ ] i ), reduced glutathione (GSH) and lipid peroxidation were investigated to evaluate the toxic effect of cocaine on hepatocytes. Cytochrome P450 induction by phenobarbital potentiated the in vitro cytotoxicity of cocaine by a factor of 13 (IC 50 = 84 μ M induced cells vs 1100 μM in non-induced cells). This difference in the susceptibility of the two types of hepatocytes to cocaine correlated well with the activity of cytochrome P450 2 B 1 2 . Rapid depletion of …

Strategies to In Vitro Assessment of Major Human CYP Enzyme Activities by Using Liquid Chromatography Tandem Mass Spectrometry

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…

New cytostatic agents obtained by molecular topology

Abstract Four new cytostatic compounds have been obtained by computer aided selection based on Molecular Connectivity, a topological approach to molecular structural study. Three of them, namely tetracycline, doxycycline and piromidic acid are well known antibiotic agents, and the fourth, carminic acid, is an innocuous colorant. Although no previous report of activity was found, on the used cell lines, for any of the selected compounds, carminic acid may be considered a new lead one since no structure-related cytostatic molecules have been reported in the literature. These results are interesting for two reasons: First, new potential low-toxicity anticancer drugs show to be a possible thera…