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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 steatosis diagnosis lack specific biomarkers. Thus, it is likely that a relevant number of children under VPA treatment may have a significant, but sub-clinical, hepatosteatosis.

Background: Hepatocyte transplantation (HT) has the potential to become a promising treatment to temporarily support liver function in patients with liver failure. Methods: 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 hepatocyte infusion. Results: The present study describes monitoring of analytical and clinical parameters and improvement of liver function following HT. In both patients, we managed to lower the blood ammonia levels and clinically improve the degree of hepatic encephalopathy, thus serving as a bridge to liver retransplantation in 1 patient. Conclusions: We believe that this therapy may be an alternative treatment in patients with chronic liver disease who suffer episodes of acute decompensation as a bridge to conventional LT.

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 protein alpha and beta (CEBP alpha and beta), peroxisome proliferator-activated receptor-gamma, coactivator 1 alpha (PGC1 alpha), and hepatocyte nuclear factor 4 alpha (HNF4 alpha)], which promoted a characteristic hepatic phenotype, as assessed by new informative markers for the step-by-step hepatic transdifferentiation of hMSC [early markers: albumin (ALB), alpha-2-macroglobuline (alpha 2M), complement protein C3 (C3), and selenoprotein PI (SEPPI) late markers: cytochrome P450 3A4 (CYP3A4), apolipoprotein E (APOE), acyl-CoA synthetase long-chain family member 1 (ACSL1), and angiotensin II receptor, type 1 (AGTR1)]. The loss of adipose adult stein cell phenotype was detected by losing expression of Thy1 and inhibitor of DNA binding 3 (Id3). The reexpression of phosphoenolpyruvate corboxykinase (PEPCK), apolipoprotein C3 (APOCIII), aldolase B (ALDOB), and cytochrome P450 1A2 (CYP1A2) was achieved by transduction with a recombinant adenovirus for HNF4 alpha and finally hepatic functionality was also assessed by analyzing specific biochemical markers. We conclude that ASCs could represent an alternative tool in clinical therapy for liver dysfunction and regenerative medicine.

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 their availability, unlimited life-span, stable phenotype, and the fact that they are easy to handle. However, the human hepatoma cells currently used (i.e. HepG2, Mz-Hep-1) show negligible levels of drug-metabolizing and do not constitute a real alternative to primary hepatocytes. Different strategies have been proposed to generate metabolically competent immortalized hepatocytes (transformation of human hepatocytes with plasmids encoding immortalizing genes, hepatocyte-like cells derived from stem cells, cell lines generated from transgenic animals, hepatocyte/hepatoma hydrid cells). Moreover, recombinant models heterologously expressing P450 enzymes in different host cells have been developed and successfully used in drug metabolism testing. In addition, new strategies have recently been explored to upregulate the expression of drug-metabolizing enzymes in cell lines of a human origin (i.e. transfection with expression vectors encoding key hepatic transcription factors). Among metabolic-based drug-drug interactions, P450 inhibition seems to be the most important. A major application of recombinant models expressing a single P450 is the screening of potential enzyme inhibitors. Therefore, pharmaceutical companies increasingly make use of cell lines to speed up the selection of new drugs with favourable pharmacokinetic and metabolic properties.

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 methodologies have been developed for the fast and routine analysis of major in vivo and in vitro CYPs enzyme activities. The high sensitivity and selectivity of mass spectrometry allow traditional assays to be minimized, thus saving time, efforts and money. Therefore this technology has become the method of choice for the fast assessment of CYP enzyme activities in early drug discovery development. Our intention herein is to review the most recent approaches that have been developed to quickly assess CYPs activities using in vitro models and liquid chromatography coupled with mass spectrometry, as well as their application in early drug discovery.

Adipose tissue represents an accessible source of mesenchymal stem cells (ADSCs), with similar characteristics to bone marrow-derived stem cells. The aim of this work was to investigate the transdifferentiation of ADSCs into hepatic lineage cells in vitro. ADSCs were obtained from human adipose tissue from lipectomy. Cells were grown in medium containing 15% AB human serum. Cultures were serum deprived for two days and exposed to a two-step protocol with two different media using growth factors and cytokines. Hepatic differentiation was assessed by RT-PCR of liver-marker genes. ADSCs exhibited a fibroblastic morphology that changed to a cuboidal shape when cells differentiated. Expression of liver genes increased when using one of the two studied media consisting of DMEM supplemented with HGF, bFGF and nicotinamide for 14 days. The results indicate that, under certain specific inducing conditions, ADSCs can be induced to differentiate into hepatic lineage in vitro. Adipose tissue may be an ideal source of high amounts of autologous stem cells.

Introduccion Tras su administracion sistemica,los adenovirus recombinantes se concentran en el higado, lo que les validaria para la transferencia genica a este organo, pero existen efectos colaterales. En este trabajo se ha analizado el impacto de la infeccion sobre la cantidad y composicion de las lipoproteinas de muy baja densidad (VLDL) secretadas en hepatocitos de rata en cultivo. Metodos Se generaron 2 vectores adenovirales, con ADNc en direccion sentido o antisentido, ambos con capacidad infectiva pero no de propagacion. Se infectaron cultivos de hepatocitos de rata con una dosis no citotoxica y se analizaron las VLDL secretadas durante los 3 periodos de 24 h posteriores a la infeccion y el contenido en lipidos del citosol. Resultados La infeccion causo un moderado descenso en la secrecion de apo B48 y de ester de colesterol durante las primeras 24 h, pero posteriormente se redujo drasticamente la secrecion de particulas con apo B48 y apo B100 y su contenido en lipidos. Ademas, modifico sus proporciones, aumentando el porcentaje de colesterol libre a costa del esterificado, que llego a ser indetectable. La menor secrecion de VLDL no conllevo una acumulacion de lipido intracelular e, incluso, descendio la masa citosolica de ester de colesterol. Conclusion La infeccion con adenovirus reduce la secrecion hepatocitaria de VLDL y su porcentaje de colesterol esterificado y, a diferencia de otros virus, estas acciones no parecen ir acompanadas de esteatosis hepatocelular.

FK506 is a potent immunosuppressive drug used for the prevention of graft rejection in organ transplantation. Experimental and clinical studies have shown correlations between apoptosis and graft rejection, and apoptosis also plays a role in cell death after ischemia-reperfusion injury in the rat liver. Fas-mediated apoptosis is very likely involved in allograft rejection and experimental evidence has shown a decrease of FasR expression in mouse hepatocytes produced by the drugs. On the basis of these findings we have investigated the protective effect of FK506 in comparison with cyclosporine A (CsA) on Fas-induced apoptosis, by analysing the activation of downstream effector caspases in human hepatocytes. Apoptosis was induced by treatment with agonistic antibodies against FasR, which resulted in a significant activation of caspase-3 after 12 h. Prevention of the downstream activation of the caspase cascade and apoptosis was observed when hepatocytes were pre-treated for 3 h with immunosuppressant drugs. A significant reduction (ca. 30-40%) of caspase-3 activation by 5 microM FK506 and CsA was observed. Along with less activation of caspase-3 a decrease of apoptotic DNA fragmentation was found. In addition, FK506 significantly reduced not only caspase-8 but also caspase-9 activation, to a similar extent as CsA, thus suggesting a protective effect at the mitochondrial level of this drug, as has already been reported for CsA. These effects of FK506 help to explain its strong anti-rejection properties and suggest promising benefits of pharmacological preconditioning on ischemia-reperfusion injury following liver transplantation.

Different types of hepatic tissue, including whole or split livers from organ donors or waste liver from therapeutic liver resections, are used to prepare human hepatocyte cultures. Characteristics of liver samples from different origins (gender, age, healthy / pathological status, xenobiotic treatment) as sources of human hepatocytes are key factors which notably determine viability and functionality of hepatocytes. The characterisation of the CYP system can be assessed in terms of activity (using specific substrates / inhibitors), protein (antibody analysis) and molecular biology-based mRNA amplification techniques (PCR technology and DNA microarrays). It could reasonably be considered that human hepatocytes reflect the heterogeneity of CYP expression in human liver and is a suitable model for drug metabolism studies. Several key issues need to be addressed at the early stages of drug development to better select drug candidates (metabolic profile and rate, identification of CYPs involved, drug-drug interactions due to enzyme induction / inhibition). The metabolic stability and metabolite profile of new chemicals can be easily investigated by incubating the drugs with fully competent metabolic models like hepatocyte suspensions or 24 h-cultured hepatocytes. CYP inhibitory effects are usually screened in recombinant CYP enzymes or microsomes, however, the actual concentration of substrate and inhibitor available to the CYP enzyme depends on processes missing in subcellular models (transport mechanisms, cytosolic enzymes, binding to intracellular proteins). Since intact cells more closely reflect the environment to which drugs are exposed in the liver, cultured hepatocytes constitute a more predictive model for drug-drug interactions. Screening of CYP inducers cannot be done in microsomes as it requires a cellular system fully capable of expressing CYP genes. Primary hepatocytes are still the unique in vitro model for global examination of inductive potential of drugs (monitored as increases in mRNA content or activity).

The possible stereoselectivity in DNA-photosensitization by carprofen (a NSAID drug) and ofloxacin (a fluoroquinolone agent) was investigated. The different drug stereoisomers or racemic mixtures were UVA-irradiated and the relaxation of the supercoiled circular pBR322 quantified by electrophoresis. Formation of single strand breaks was compared for each group of compounds. Moreover a mechanistic study by means of repair enzymes: T4 endonuclease V (specific of cyclobutane pyrimidine dimers), E. coli endonuclease III (revealing oxidized pyrimidines) and E. coli Formamidopyrimidine-DNA glycosylase (revealing oxidized purines) provided further insights into a possible stereoselectivity of the different reaction pathways in drug photosensitized-DNA damage. Ofloxacin and levofloxacin (its S stereoisomer) were responsible of single strand breaks formation as well as oxidation of pyrimidine and purine bases. No pyrimidine dimers were observed. Racemic, R and S stereoisomers of carprofen were less efficient than ofloxacin in DNA single strand breaks formation and did not induce enzyme-sensitive sites. The photoproducts distribution of drug-photosensitized reactions of 2'-deoxyguanosine and thymidine were established by HPLC as fingerprints for assignment of the DNA-photosensitization mechanism. Both Type I and Type II mechanisms were assigned to nucleoside-photosensitization by ofloxacin and levofloxacin. In the case of carprofen, a weak nucleoside degradation was obtained. The data suggest that levofloxacin, the (S) stereoisomer, might be slightly more efficient than racemic ofloxacin. In the case of carprofen the (S) isomer appears to be somewhat less active than its (R) enantiomer. However, due to the small differences found, the possible stereoselectivity has to be confirmed by future studies.

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 vector. The relevance of these findings was further explored in the more complex DNA/chromatin structure within endogenous CYP3A4 gene. Using appropriate adenoviral expression vectors, we found that both hepatic and nonhepatic cells overexpressing C/EBP alpha had increased CYP3A4 mRNA levels, but no effect was observed when HNF-3 gamma was overexpressed. In contrast, overexpression of HNF-3 gamma simultaneously with C/EBP alpha resulted in a greater activation of the CYP3A4 gene. This cooperative effect was hepatic-specific and also occurred in CYP3A5 and CYP3A7 genes. To investigate the mechanism for HNF-3 gamma action, we studied its binding to CYP3A4 promoter and the effect of the deacetylase inhibitor trichostatin A. HNF-3 gamma was able to bind CYP3A4 promoter at a distal position, near the most distal C/EBP alpha binding site. Trichostatin A increased C/EBP alpha effect but abolished HNF-3 gamma cooperative action. These findings revealed that C/EBP alpha and HNF-3 gamma cooperatively regulate CYP3A4 expression in hepatic cells by a mechanism that probably involves chromatin remodeling.

c-Myc transcription factor is a key protein involved in cellular growth, proliferation and metabolism. c-Myc is one of the most frequently activated oncogenes, highlighting the need to identify intracellular molecules that interact directly with c-Myc to suppress its function. Here we show that Hhex is able to interact with the basic region/helix-loop-helix/leucine zipper of c-Myc. Knockdown of Hhex increases proliferation rate in hepatocellular carcinoma cells, whereas Hhex expression cell-autonomously reduces cell proliferation rate in multiple cell lines by increasing G1 phase length through a c-Myc-dependent mechanism. Global transcriptomic analysis shows that Hhex counter-regulates multiple c-Myc targets involved in cell proliferation and metabolism. Concomitantly, Hhex expression leads to reduced cell size, lower levels of cellular RNA, downregulation of metabolism-related genes, decreased sensitivity to methotrexate and severe reduction in the ability to form tumours in nude mouse xenografts, all indicative of decreased c-Myc activity. Our data suggest that Hhex is a novel regulator of c-Myc function that limits c-Myc activity in transformed cells.

1. The role of endogenous nitric oxide in rat hepatocyte functionality and survival in cell culture was examined. Towards this aim, cytochrome P450 activities (CYP1A1/2, 2B1, 2A1, 2C11, 2D1, 2E1 and 3A1), liver-specific metabolic functions and cell survival were comparatively evaluated in hepatocytes isolated from the male Sprague-Dawley rat and/or cultured in control conditions or in the presence of N-omega-nitro-L-arginine methyl ester (NAME), an inhibitor of nitric oxide synthesis. 2. Suppression of nitric oxide production by NAME paralleled a substantial preservation of hepatocyte phenotype in culture. The presence of NAME was particularly important during isolation and/or the 6-24h culture. By 24h, beneficial effects were evident in parameters particularly unstable in culture (glycogen content, P450), whereas no changes were produced in well-preserved functions (glucose, urea and albumin synthesis, glutathione, drug-conjugating enzymes). 3. Long-term treatment of hepatocytes with NAME also produced a reduction in caspase 3 activation and in the percentage of spontaneous apoptotic cells, and an increase in cell survival and transcriptional activity as shown by attached cellular protein content and the protein-DNA ratio respectively. 4. In conclusion, inhibition of early endogenous nitric oxide formation is an efficient procedure for obtaining hepatocyte cultures with stable expression of differentiated functions, high cell survival and few signs of cell senescence.

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 and Western blot analysis. The functionality of each expressed CYP2C was assessed by determining specific catalytic activities in these cells, that is, taxol-6-hydroxylation for CYP2C8; diclofenac-4'-hydroxylation for CYP2C9; S-mephenytoin-4'-hydroxylation for CYP2C18; S-mephenytoin-4'-hydroxylation for CYP2C19. As a result of the combined strategies used here, the transfected cells showed activities four to seven times higher than those of 24-hour cultured hepatocytes.

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 photosensitizing drugs gave rise to similar processes when irradiated in the presence of BSA or the isolated amino acids. In conclusion, histidine and tyrosine appear to be key sites for the photosensitized damage to proteins. Photodegradation of the isolated amino acids in vitro may be an indicator of the photosensitizing potential of drugs.

Resumen: El trasplante hepático (TH) es el único tratamiento efectivo existente para las enfermedades hepáticas en fase terminal. La desproporción entre demanda y oferta de órganos constituye su principal limitación, planteando la necesidad de buscar alternativas de tratamiento.El trasplante de hepatocitos humanos o trasplante celular hepático (TCH) constituye, en el momento actual, la mejor opción terapéutica puente al restablecimiento de la función hepática o al trasplante hepático. Consiste en trasplantar hepatocitos humanos totalmente diferenciados a un órgano receptor, en cantidad suficiente para que estos sobrevivan y restauren la función hepática normal, basándose en la capacidad de regeneración hepática.El TCH consta básicamente de 4 pasos: el aislamiento de los hepatocitos a partir de injertos hepáticos descartados para TH, la preparación de las suspensiones celulares, la criopreservación de los hepatocitos aislados y, finalmente, su implante en el receptor.Esta terapia se ha llevado a cabo en pacientes con insuficiencia hepática aguda de distintas etiologías con intención de sustituir o servir de puente al TH y en el tratamiento de pacientes pediátricos con errores congénitos del metabolismo con objetivo de reemplazar el déficit enzimático causante de la enfermedad.En el Hospital La Fe de Valencia hemos puesto en marcha una Unidad de Terapia Celular Hepática y llevado a cabo el primer TCH en España, abriendo una nueva línea de trabajo dentro del Programa de Trasplante Hepático. Abstract: Liver transplantation has been remarkably effective in the treatment of patients with end-stage liver disease. However, disparity between solid-organ supply and increased demand is the main limitation, resulting in longer waiting times and an increase in the mortality of transplant recipients. This situation creates the need to seek alternatives to orthotopic liver transplantation. Hepatocyte transplantation or liver cell transplantation has been proposed as the best method to support patients, a bridge to restore liver function or liver transplant.The procedure consists in transplanting individual cells in a recipient organ in enough quantity to survive and restore the function. The capacity of hepatic regeneration constitutes the biological basis of hepatocyte transplantation. Liver cell transplantation is carried out by means of the isolation of hepatocytes from donor liver rejected for orthotopic transplantation, to prepare a cell suspension for infusion, cryopreservation and, finally, hepatocytes are implanted into the recipient.This may be an optional therapeutic procedure in some patients with inborn errors of metabolism, fulminant hepatic failure, and acute and chronic liver failure, as a bridge to orthotopic liver transplantation.The first hepatocyte transplantation in Spain was performed in the Cell Therapy Unit of the Hospital La Fe of Valencia, creating a new research line in the transplant program.

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 therapy, and second, the usefulness of our topological approach in evaluating pharmacological mechanisms of action, already demonstrated in the search for other therapeutical agents, is confirmed.

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 method are its simplicity and automation, the reduction in sample handling, and the small number of cells required compared to other conventional methods.

The various enantiomers of ketoprofen (S and R) and its racemic form ( R S ) exhibited comparable phototoxicities when examined by the following in vitro test systems: (a) effects of pre-irradiated drugs on cultured hepatocytes; (b) co-irradiation of drugs with hepatocytes or fibroblasts; (c) photohaemolysis sensitized by the various ketoprofen steroisomers; (d) drug-photosensitized formation of linoleic acid hydroperoxides. Inhibition of photohaemolysis and photodynamic lipid peroxidation by butylated hydroxyanisole and reduced glutathione suggests that the phototoxicity of ketoprofen is associated with a radical chain (type I) peroxidation of membrane lipids, leading to cell lysis In view of the above results it could be advantageous to use the pharmacologically active S(+) enantiomer instead of the R/S form, since the lower doses required would result in a diminished phototoxic potential.

The microfluorometric assay, suitable for measuring microsomal epoxide hydrolase activity in cultured cells, is based on the conversion of benzo[ a ]pyrene-7,8-epoxide to the corresponding trans -benzo[ a ]pyrene-7,8-dihydrodiol, a compound that fluoresces at 403 nm when excited at 365 nm. Activity is determined by incubating S9 fractions obtained from microcultures with this epoxide and monitoring the fluorescence with a microplate reader. Under the assay conditions selected, the photodecomposition of the reaction product was minimized and the linearity of the reaction was extended. The major advantages of this method are: (1) high sensitivity with a detection limit of 5 pmol/well of trans -benzo[ a ]pyrene-7,8-dihydrodiol formed, which is comparable to the most sensitive radioactive methods; (2) minimal sample requirement (1-5 μg liver microsomes; 10-50 μg S9 fraction from cultured cells); (3) reduced consumption of hazardous reagents; and (4) a considerable reduction in assay time and facility for simultaneous determination of enzyme activity in multiple samples.

The photobinding of drugs to cells has been investigated as a possible indicator of the photoallergic potential. Tiaprofenic acid (TA), carprofen (CP), benoxaprofen (BP) and ibuprofen (IP) were selected as test substances and human fibroblasts as model biological system. Radioactive labelling of the drugs was achieved through alkaline exchange of the α-carboxyl hydrogens by tritium, using 3H2O as solvent. When the labelled compounds were co-irradiated with fibroblasts, TA gave rise to the highest amount of radioactivity covalently bound to cells. This also occurred, albeit to a lower extent, with CP and BP; by contrast, no effect was observed for IP. The observed rank order is in agreement with the available in vivo data. The time course of the process was determined for TA, distinguishing between covalent and non-covalent binding. The results, together with gas Chromatographie analyses of the irradiation mixtures and binding studies with TA and its major photoproduct decarboxytiaprofenic acid (decarboxy-TA) in the dark, indicated that most of the observed photobinding might be due to the photoproduct rather than to the parent drug. It is conceivable that hydrogen abstraction by the excited ketone could take place with proteins as reaction partners. This process would lead to the generation of a radical pair, whose coupling would result in the formation of a covalent bond. The contribution of this mechanistic pathway to the in vivo photobinding has to be considered when a lipophilic photoproduct is formed which, as for decarboxy-TA, still contains an active chromophore.

Environmental Safety Laboratory, Unilever Research, Colworth House, Sharnbrook,Bedford MK44 1LQ, UK; Unidad de Hepatologia Experimental, Hospital Universitario La Fe, Avda de Campanar 21, 46009 Valencia, Spain; FRAME, Russell & Burch House, 96-98 North Sherwood Street, Nottingham NG1 4EE, UK; School of Pharmacy, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF, UK; ECVAM, JRC Environment Institute, 21020 Ispra (Va), Italy; Bundesinstitut fur gesundheitlichen Verbraucherschutz und Veterinarmedizin (BgVV), Thielallee 88-92,14195 Berlin, Germany; Istitutodi R icercasulla Senescenza, Sigma-Tau, Via Pontina, km 30.400, 00040 Pomezia, Italy; Sanofi Research Division, Alnwick Research Centre, Alnwick, NorthumberlandNE66 2 JH, UK;ChemQuest, Cheyney House, 19-21 Cheyney Street, Steeple Morden, Herts. SG8 OLP, UK; ZENECA Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SKI 0 4TJ, UK; TNO Nutrition and Food Research Institute, Division of Toxicology, P.O. Box 360, 3700 AJ Zeist, The Netherlands; Research Institute of Toxicology (RITOX), Utrecht University, P.O. Box 80.176, Yalelaan 2, 3508 TD Utrecht, The Netherlands; SIAT, Missionsstrasse 60, 4055 Basel, Switzerland

No consensus exists on how to address possible toxicity of nanomaterials as they interfere with most in vitro screening tests based on colorimetric and fluorimetric probes such as the dichloro-dihydrofluorescein diacetate (DCFH-DA) assay for detection of oxidative species. In the present research, nanomaterial interaction with DCFH-DA was studied in relation to its nature and/or assay conditions (cell-based and time exposure) by incubating Rhodamine (Rhd)-labeled 25 nm and 50 nm silica (SiO2), naked and oleic acid coated magnetite, (Fe3O4) and maghemite (Fe2O3) iron oxide, titanium dioxide (TiO2) and poly(ethylene oxide)-poly(lactide/glycolide) acid (PLGA-PEO) nanoparticles (NPs) with metabolically active rat hepatocytes for 4 and 24-h periods. Data indicated that nanoparticle uptake correlated with quenching of dye fluorescence emission. In spite of their masking effect, the oxidative potential of NPs could be detected at a limited threshold concentration when exposed for periods of time longer than those frequently used for this test. However, changes in the experimental conditions did not systematically result in free radical formation for all nanomaterials tested. Overall data indicate that despite the quenching effect of nanoparticles on DCFH-DA assay, it can be considered as a useful tool for quantitative measurement of NPs-induced oxidative stress by minor modifications of standardized protocols.

The quantification of P -450 enzymes (CYP proteins) is of great relevance for drug metabolism and toxicity studies. Their measurement in cultured cells requires a sufficient amount of cellular protein to isolate a minimal amount of functional microsomes. The difficulties in obtaining microsomes from a small number of cultured hepatocytes make it necessary to use more sensitive methods. Immunological methods can overcome such drawbacks. However, their use is prevented by the fact that P -450 enzymes are membrane proteins that require the use of detergents for adequate solubilization which, in turn, can interfere with the antigen-antibody recognition. A sensitive immunoassay (competitive indirect ELISA) was developed which allowed quantification of CYP1A1 and CYP2B1/2 in small culture plates with a reduced number of cells. The main advantages of this ELISA were: (1) precise measurement of both proteins over a wide range of antigen concentration (10–10,000 fmol CYP proteins); (2) minimal sample handling allowing reproducible titrations in small number of cells; (3) minimization of interference by detergents; (4) an excellent correlation (r = 0.96–0.98) between the immunologically determined CYP1A1 and CYP2B1/2, and the activities of ethoxyresorufin- O -deethylase (EROD) and pentoxyresorufin- O -depentylase (PROD), respectively. This assay allowed a precise monitoring of the expression of CYP proteins in cultured cells. Upon incubation of hepatocytes with 3-methylcholanthrene the levels of immunoreactive CYP1A1 and CYP2B1/2 were paralleled by an increase of the enzymatic activities of EROD and PROD, respectively. In cultured rat hepatocytes treated with phenobarbital, significant EROD activity was detected in the absence of immunoreactive CYP1A1 induction. Analogously, there was a dissimilarity between enzymatic activity and protein levels in ageing cultures: the decrease of PROD activity preceded by several hours that of the immunoreactive CYP2B1/2, which emphasizes the need to determine individual CYPs, in addition to their enzymatic activities.

The photochemical behaviour of 2-arylpropionic acids involves the intermediacy of short-lived species such as drug-derived peroxides and/or free radicals. Although peroxides are expected to undergo very rapid breakdown, this study shows that they can be detected in the photomixtures by the peroxidase-catalysed oxidation of dichlorodihydrofluorescein to its fluorescent analogue. Their possible contribution to phototoxicity has been evaluated by red blood cell membrane lysis. The shorter lives of organic free radicals prevent their direct determination in the irradiation mixtures; however, the results strongly suggest that these intermediates have a key role in the phototoxicity of 2-arylpropionic acids, through induction of photodynamic lipid peroxidation. This has been determined using linoleic acid as a model lipid, and measuring the spectrophotometric absorption at 233 nm associated with the formation of dienic hydroperoxides. It is probable that a similar process is responsible for the photochemically induced damage to cell membranes observed in the photohaemolysis assays.

The effects of human recombinant interferons alpha and gamma on monooxygenase activities in cultured human hepatocytes have been investigated. Dose-response and time course studies showed that interferons reduced 7-ethoxyresorufin O-deethylase activity of human hepatocytes after a 12-hr incubation with 300 U/ml interferons alpha and gamma (52% and 38% decrease, respectively). A reduction in 7-ethoxyresorufin O-deethylase activity was also observed in HepG2 cells, although in these cells maximal inhibition was observed after 24 hr of treatment with 1000 U/ml (a 41% and 28% decrease with interferon alpha and gamma, respectively). A decrease in activity was also observed in 7-pentoxyresorufin O-depentylase, 7-ethoxycoumarin O-deethylase and testosterone 2alpha- and 6beta-hydroxylase. It is noteworthy that the marked increase in 7-ethoxyresorufin O-deethylase activity detected in human hepatocytes after incubation with 2 mum-3-methylcholanthrene (10-fold over non-treated cells) was reduced by 40% in the presence of interferons (300 U/ml), thus indicating that the inducibility of monooxygenases could be altered by interferon treatment. The inhibitory effect of interferons on 7-ethoxyresorufin O-deethylase was transient and HepG2 cells recovered their normal activity 24 hr after interferon removal from culture medium. This study provides the first direct evidence that interferons down-regulate the level of monooxygenases in human hepatic cells and prevent, in part, their induction by xenobiotics.

The present study deals with the expression of cytochrome P-450 and two monooxygenase activities (aryl hydrocarbon hydroxylation and 7-ethoxycoumarin O-deethylation) by hepatocytes cultured on feeder layers of MS cells, in an attempt to investigate the influence of culture medium composition on phase I activities of hepatocytes. When compared with culture medium I (serum-free, hormone-supplemented Ham's F12), 4-day MS hepatocyte cultures supplemented with nicotinamide (medium II) allowed better retention by hepatocytes of cytochrome P-450 and phase I activities (aryl hydrocarbon hydroxylation and 7-ethoxycoumarin O-deethylation). A moderate additive effect was found after increasing branched-chain amino acid composition (medium III). Increasing the concentration of all amino acids, galactose and pyruvate (medium IV) resulted in higher levels of cytochrome P-450, aryl hydrocarbon hydroxylation and 7-ethoxycoumarin O-deethylation which, after 8 days in culture, were at 33, 49 and 43% of day 0 values, respectively. A further increase of branched-chain amino acid concentration produced only a moderate effect (medium V). Under these culture conditions, hepatocytes also expressed basic metabolic functions. After 10 days, MS hepatocyte cultures retained 43, 71 and 65% of ureogenesis, triglyceride secretion and albumin production measured at day 0. In contrast, pure hepatocytes cultured under the same conditions expressed these three hepatic functions only until the fifth or sixth day of culture.

The liver is the most important target for toxicity caused by drugs. This vulnerability is a consequence of the functional features of the liver and their role in the metabolic elimination of most drugs. Therefore, evaluation of potential hepatotoxicity represents a critical step in the development of new drugs. The liver is very active in metabolising foreign compounds and, although biotransformation reactions generally parallel detoxification processes, the formation of reactive metabolites is relatively frequent. Thus, drug-induced hepatotoxicity can be due to the administered compound itself or to metabolites formed by hepatic metabolism. The most important systems to study hepatotoxicity and metabolic activity in vitro are liver slices, isolated liver cells in suspensions or in primary cultures including co-culture methods and special 3D techniques, various subcellular fractions and hepatic cell lines. These models can be used for cytotoxicity and genotoxicity screening, and also to identify the mechanisms involved in drug-induced hepatotoxicity. Assessment of current cytotoxicity and hepatic-specific biochemical effects are limited by the inability to measure a wide spectrum of potential mechanistic changes involved in the drug-induced toxic injury. A convenient selection of end-points allows a multiparametric evaluation of drug toxicity. In this regard, omic (cytomic, metabonomic, proteomic and toxicogemic) approaches help defining patterns of hepatotoxicity for early identification of potential adverse effects of the drug to the liver. The development of robust in vitro-based multiparametric screening assays covering a wider spectrum of key effects will heighten the predictive capacity for human hepatotoxicity, and accelerate the drug development process.

In this work, the phototoxicity of two non-steroidal anti-inflammatory drugs, Butibufen and Flurbiprofen, was examined. Both were unstable to light, to give several photoproducts which were isolated and identified. The different photoproducts were formed by a primary photochemical mechanism which involves an initial cleavage of the CC bond α to the carbonyl group, followed by several secondary processes. The cytotoxic effects of the xenobiotics were evaluated using two well-established biological in vitro tests: (a) enzyme leakage lactate dehydrogenase and glutamate-oxaloacetate transaminase from cultured fibroblasts and (b) lysis of red blood cells. The benzylic alcohols caused extensive leakage from cultured fibroblasts at the different concentrations assayed. The alcohol obtained from Butibufen was a potent lytic agent for human red blood cells. The other photoproducts, Butibufen and Flurbiprofen did not produce observable toxic effects on cells.

The cytotoxic effect of cocaine on human hepatocytes was evaluated after 24 hr of exposure to cocaine by measuring the leakage of intracellular lactate dehydrogenase and the reduction of MTT. According to these endpoint parameters, the half-maximal cytotoxic concentration (IC 50 ) of cocaine was 6.8 and 7.8 m m , respectively. Lower concentrations of cocaine, however, impaired basic metabolic functions of human hepatocytes. Exposure of cells to 2 m m -cocaine resulted in a 50% decrease in hepatic glycogen, a 40% decrease in cellular glutathione content and a 40% decrease in urea synthesis with respect to control values. Ethanol greatly potentiated cocaine-induced hepatotoxicity. After a 48-hr pretreatment of human hepatocytes with 50 m m -ethanol, concentrations of cocaine (0.25 m m ) that had no effects on hepatocyte metabolism in the absence of ethanol, caused a 20% inhibition of the urea synthesis rate, a 40% depletion of glycogen stores and a 30% reduction in glutathione content. The results of our work show that ethanol significantly increases the toxic effects of cocaine on human hepatocytes.

This study examines the ability of externally added S-adenosyl-L-methionine (SAMe) to act as a precursor of intracellular glutathione (GSH) and to prevent the GSH depletion induced by paracetamol, opiates and ethanol in rat and human hepatocytes.

1. The hepatotoxic effects of heroin and methadone, and the effect of ethanol on opioid-induced hepatotoxicity, have been investigated in human cultured hepatocytes. Hepatocytes pretreated with 50 and 100 mM ethanol were exposed to increasing concentrations of heroin and methadone. 2. Cytotoxicity was evaluated by measuring leakage of intracellular lactate dehydrogenase, and by assessment of hepatocyte mitochondrial succinate dehydrogenase. The half-maximal cytotoxic concentration of heroin for human hepatocytes (TC50) was decreased by 70-55% by pre-exposure to 50 mM ethanol, and that for methadone was decreased by 60-40%. 3. Metabolic functions of human hepatocytes were significantly impaired at concentrations of opioids that had shown little cytotoxicity. Ethanol potentiated opioid-induced hepatotoxicity; concentrations of heroin and methadone that had little or no effect on hepatocyte metabolism in the absence of ethanol caused a significant decrease in urea synthesis rate, metabolism of glycogen and depletion of the intracellular GSH pool after ethanol pretreatment. 4. The increase in toxicity of heroin and methadone produced by ethanol is concomitant with a 40% increase in cytochrome P-450 levels of the pretreated hepatocytes.

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 hepatocytes. Moreover, the evaluation of urea synthesis from ammonia and UDP-glucuronosyltransferase 1A1 activity, a newly developed assay using beta-estradiol as substrate, allows the possibility of customizing cell preparation for receptors with urea cycle disorders or Crigler-Najjar Syndrome type I. Sources of human liver and factors derived from the procurement of the liver sample (warm and cold ischemia) have also been investigated. The results show that grafts with a cold ischemia time exceeding 15 h and steatosis should not be accepted for hepatocyte transplantation. Finally, livers from non-heart-beating donors are apparently a potential suitable source of hepatocytes, which could enlarge the liver donor pool.

This study provides the first experimental evidence of the short-term control of fructose 2,6-bisphosphate (Fru(2,6) P 2 ) levels in adult human hepatocytes. (1) In hepatocytes whose metabolic status resembles the fed state (glycogen-rich), exposure to glucagon (10 −8 M) caused a drastic decrease in the levels of this effector and a significant fall in lactate production rate. Adrenaline, isoprenaline (a β-adrenergic agonist) and lactate exerted a similar action decreasing Fru(2,6) P 2 concentration. (2) In glucagon pre-treated, glycogen- and Fru(2,6) P 2 -depleted cells (a situation that mimics the fasted state), Fru(2,6) P 2 re-synthesis was strictly dependent on glucose availability. (3) Insulin did not seem to exert a direct action on the control of Fru(2,6) P 2 in human hepatocytes. The hormone - which failed to enhance Fru(2,6) P 2 in glucose-starved cells - did not further increase Fru(2,6) P 2 content nor its time-course evolution as compared to hepatocytes incubated with glucose alone. (4) Lactate caused a significant delay in the glucose-induced increase in Fru(2,6) P 2 content that vould not be prevented by insulin. (5) Data indicate that in human hepatocytes glucose is a more powerful modulator of Fru(2,6) P 2 than insulin, and that variations in blood lactate concentration may also play a role in the control of hepatic Fru(2,6) P 2 levels during the fasted-to-fed transition in humans.

The activity of human cytochrome P450 monooxygenases, aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O -deethylase can be increased by 3-methylcholanthrene, phenobarbital and ethanol in human hepatocytes maintained in primary culture. Total cytochrome P450 content increased two-fold after 48 hr of incubation with methylcholanthrene or phenobarbital and 1.5-fold after incubation with ethanol. The three chemicals elicited different effects on cytochrome P450 dependent activities. Addition of 3-methylcholanthrene caused a time- and concentration-dependent increase in both mono-oxygenase activities, aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O -deethylase, while phenobarbital and ethanol increased 7-ethoxycoumarin O -deethylase activity but had no effect on aryl hydrocarbon hydroxylase. Dexamethasone per se had little or no effect on either monooxygenase activities, but potentiated the effect of the three chemicals on 7-ethoxycoumarin O -deethylase.

Co-culture of hepatocytes with fibroblastic cells (C3H/10T 1 2 and 3T3) allowed the expression of phase I and II biotransformation enzymes for a prolonged time in culture. After 7 days in co-culture, cytochrome P-450 and monooxygenase activities were still 20–30% of the initial value, while in pure hepatocyte cultures they were undetectable. NADPH-cytochrome c reductase and conjugation activities remained at nearly initial levels for at least 7 days in co-cultured hepatocytes. Finally, in co-cultures a clear and prolonged induction of monooxygenase activities by 3-methylcholanthrene and phenobarbital was observed.

The multicentre evaluation study of in vitro cytotoxicity tests (MEIC) is organized by the Scandinavian Society of Cell Toxicology. All interested laboratories are invited to test a published list of 50 reference chemicals in their various in vitro assays with a bearing on general toxicity. Submitted results will be centrally evaluated for their relevance to human toxicity, including a comparison with the efficiency of conventional animal tests. This brief communication presents the very first preliminary results of the study, that is, prediction of human acute lethal toxicity for the first 10 MEIC chemicals by all the results submitted to date, that is, five in vitro cytotoxicity assays. As a baseline for judging the efficiency of the cytotoxicity tests, rat and mouse LD 50 values were compared with human acute lethal dosage of the chemicals. Rat LD 50 prediction was relatively poor, but mouse LD 50 values correctly predicted the human lethal dose for six out of the 10 substances. A multivariate method of comparison including all cytotoxicity test results, predicted human lethal blood concentrations as well as the mouse LD 50 prediction of dosage. Since the blood concentrations used in the comparison were derived from human lethal dosage with the help of two simple pharmacokinetic factors (absorbed fraction in the intestine and distribution volume of chemicals), the cytotoxicity assays were found also to be able to predict human dosage, as well as did the mouse LD 50 prediction.

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 microg/mL), followed by T. testudinum and Erythroxylum extracts, whereas no significant protective effects was produced by Dictyota extract treatment. Antioxidant properties of the natural products against lipid peroxidation and GSH depletion induced by tert-butyl hydroperoxide were then investigated. The results show that at 36 h pre-treatment of cells with mangiferin or MSBE, concentrations of T. testudinum and Erythroxylum extracts ranging from 25 to 100 microg/mL significantly inhibited lipid peroxidation induced by tert-butyl hydroperoxide (100 and 250 microM) and increased the GSH levels reduced by the toxicant. D. pinnatifida inhibited lipid peroxidation, but did not preserve GSH levels. In conclusion, MSBE, E. minutifolium, E. confusum and T. testudinum extracts and mangiferin showed hepatoprotective activity against induced damage in all the experimental series, where mangiferin and the extracts of MSBE and T. testudinum were the best candidates to inhibit "in vitro" damage to rat hepatocytes. This hepatoprotective effect found could be associated with the antioxidant properties observed for the products.

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 only moderate effects were induced by 1000 microg/mL Tt. Seven specific P450 activities were evaluated after 48 h exposure of cells to the products. MSBE reduced phenacetin O-deethylation (POD; CYP1A2) activity in a concentration-dependent manner (IC(50)=190 microg/mL). No decreases were observed in other activities. In contrast, mangiferin produced reductions in five P450 activities: IC(50) values of 132, 194,200, 151 and 137 microg/ml for POD (CYP1A2), midazolam 1'-hydroxylation (M1OH; CYP3A1), diclofenac 4'-hydroxylation (D4OH; CYP2C6), S-mephenytoin 4'-hydroxylation (SM4OH), and chlorzoxazone 6-hydroxyaltion (C6OH; CYP2E1), respectively. E. minutifolium, E. confusum and Tt extracts produced small reductions in SM4OH and C6OH activities, but no significant changes were noted in the other P450 activities. On the other hand, all the products increased the benzyloxyresorufin O-debenzylation (BROD; CYP2B1) activity, with MSBE, mangiferin or E. minutifolium showing the highest effects (about 2-fold over control). Our results showed in vitro effects of these natural products on P450 systems, possibly leading to potential metabolic-based interactions.

The study of the ADME features of the huge number of new chemical entities (NCEs) produced mainly by combinatorial chemistry has become a bottleneck in the drug development process. In response the pharmaceutical industry is involved in the development of new medium/high-throughput screening capabilities. The aim of this paper is to review some of the available in vitro ADME systems adapted to screening requirements together with the technological approaches which can be linked to medium/high-throughput molecular screening.

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 spectrometry has allowed traditional assays to be minimized, thus enabling the use of 96-well plate format which markedly reduced the number of hepatocytes needed for each cytochrome CYP450 activity measurement, a fact that is particularly critical concerning human hepatocytes. (C) 2007 Elsevier Ltd. All rights reserved.

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 accumulation of lipids in the cytosol. Decreased ECOD and testosterone oxidation were found after 14 h of exposure to 1 mM or 2 mM FFA (about 60–70% and 30–60% of control, respectively). The effects of fat-overloading on individual P450s were analyzed both at activity and mRNA level. CYP1A2, CYP2C9, CYP2E1 and CYP3A4 activities were reduced after hepatocyte incubation with 1 mM (to 45–65% of control) or 2 mM (to 20–50%) FFA for 14 h. Reductions in P450 transcripts were also found in hepatocytes treated with 1 mM FFA. Our findings showed a general down-regulation of P450s involved in drug metabolism in fat-overloaded hepatocytes. The results suggest that, despite their reduced P450 function, human hepatocytes obtained from donors with steatosis are metabolically competent and could be used for drug metabolism studies.

The extract of the fern Polypodium leucotomos (PL, Fernblock (R)) is an oral photoprotectant with strong antioxidative properties. Recent studies to determine its chemical composition have shown 4-hydroxycinnamic acid (p-coumaric), 3 methoxy-4-hydroxycinnamic acid (ferulic), 3,4-dihydroxycinnamic acid (caffeic), 3-methoxy-4-hydroxybenzoic acid (vanillic) and 3-caffeoilquinic acid (chlorogenic) to be among its major phenolic components. No conclusive data are available, however, on the H2O2-scavenging capacity of these compounds, or on their absorption and metabolism following their oral intake. In the present work, their antioxidative capacity was assessed by the luminol/H2O2 assay, their absorption studied using Caco-2 cells to resemble the intestinal barrier, and their metabolism investigated using cultured primary rat hepatocytes. The antioxidant capacity of PL components increased in a concentration-dependent manner, with ferulic and caffeic acids the most powerful antioxidants. The apparent permeability results correspond to a human post-oral administration absorption of 70-100% for all tested substances. Coumaric, ferulic and vanillic acids were metabolized by CYP450-dependent mono-oxygenases and partially conjugated to glucuronic acid and sulfate. These phenolic compounds may contribute to the health benefits afforded by this oral photoprotectant. (c) 2005 Elsevier Ltd. All rights reserved.

The major possible sources of human liver for hepatocyte isolation are elective liver biopsies, cadaveric liver grafts and therapeutic liver resections. The suitability in terms of metabolic-competent hepatocyte cultures and risk/benefit of these resources has been comparatively studied. To this end, viability of isolated hepatocytes, yield of isolation procedure, hepatocyte survival during culture and CYP activities were the parameters analysed. The best results were found in hepatocytes prepared from elective biopsies, whereas a marked reduction in viability and functional competence was seen in hepatocytes from hepatectomy samples. Metabolic differences were observed in total CYP oxidative metabolism (7-ethoxycoumarin O-deethylation, total testosterone hydroxylation), as well as in CYP3A4, CYP2C9 or CYP2C19 activities (testosterone oxidations at 6beta-, 16beta- and 17-positions, respectively). Vascular control during the hepatectomy procedure influenced hepatocyte functionality: higher CYP activities were found in hepatocytes isolated from samples obtained under non-ischemic conditions or continuous vascular clamping than in those obtained under intermittent vascular clamping. In addition to cellular functionality, other criteria such as sample availability or ethical aspects should be considered. Elective biopsies have low, but not absent, surgical risk. However, the better functionality and the higher accessibility of elective liver samples in comparison to the other groups suggest this source of liver tissue as the most appropriate for cell harvesting purposes.

Drugs are usually biotransformed into new chemical species that may have either toxic or therapeutic effects. Drug metabolism studies are routinely performed in laboratory animals but, due to metabolic interspecies differences when compared to man, they are not accurate enough to anticipate the metabolic profile of a drug in humans. Human hepatocytes in primary culture provide the closest in vitro model to human liver and the only model that can produce a metabolic profile of a given drug that is very similar to that found in vivo. However their availability is limited due to the restricted access to suitable tissue samples. The scarcity of human liver has led to optimising the cryopreservation of adult hepatocytes for long-term storage and regular supply. Human hepatocytes in primary culture express typical hepatic functions and express drug metabolising enzymes. Moreover, qualitative and quantitative similarities between in vitro and in vivo metabolism of drugs were observed. Different strategies have been envisaged to prolong cell survival and delay the spontaneous decay of the differentiated phenotype during culture. Thus, hepatocytes represent the most appropriate model for the evaluation of integrated drug metabolism, toxicity/metabolism correlations, mechanisms of hepatotoxicity, and the interactions (inhibition and induction) of xenobiotics and drug-metabolising enzymes. However, in view of limitations of primary hepatocytes, efforts are made to develop alternative cellular models (i.e. metabolic competent CYP-engineered cells stably expressing individual CYPs and transient expression of CYPs by transduction of hepatoma cells with recombinant adenoviruses). In summary, several cellular tools are available to address key issues at the earliest stages of drug development for a better candidate selection and hepatotoxicity risk assessment.

Photobinding of drugs to biomolecules constitutes an important early event in the onset of photoallergy. In the present work, UV irradiation of human serum albumin in the presence of either suprofen (SUP) or its major photoproduct, decarboxylated suprofen (DSUP), has been studied as a model system for drug-photosensitised protein binding. Both dark binding and binding in the presence of light were investigated since this will affect the mode, site and mechanism of drug interaction with the protein. In order to determine the binding features of SUP to albumin, competitive binding experiments were carried out using fluorescent probes specific for site I and II. Suprofen was found to selectively dark bind to site II on HSA. Photobinding of DSUP to HSA was more efficient than SUP. Parallel to this, the intrinsic tryptophan fluorescence of HSA decreased when the protein was previously irradiated in the presence of the photoactive compounds, again being DSUP more efficient compared with SUP. As fluorescence quenching involves electron transfer from the excited Trp to the ground state DSUP, it follows that the photoactive compound binding to HSA must be on (or in close proximity to) site I Trp(214) residue. It appears that photobinding of SUP is largely preceded by its photodecomposition to DSUP which, in turn, associates and photobinds to HSA. (C) 2001 Elsevier Science Ltd. All rights reserved.