RESUMEN
The assessment of the carcinogenic potential of chemicals with alternative, human-based in vitro systems has become a major goal of toxicogenomics. The central read-out of these assays is the transcriptome, and while many studies exist that explored the gene expression responses of such systems, reports on robustness and reproducibility, when testing them independently in different laboratories, are still uncommon. Furthermore, there is limited knowledge about variability induced by the data analysis protocols. We have conducted an inter-laboratory study for testing chemical carcinogenicity evaluating two human in vitro assays: hepatoma-derived cells and hTERT-immortalized renal proximal tubule epithelial cells, representing liver and kidney as major target organs. Cellular systems were initially challenged with thirty compounds, genome-wide gene expression was measured with microarrays, and hazard classifiers were built from this training set. Subsequently, each system was independently established in three different laboratories, and gene expression measurements were conducted using anonymized compounds. Data analysis was performed independently by two separate groups applying different protocols for the assessment of inter-laboratory reproducibility and for the prediction of carcinogenic hazard. As a result, both workflows came to very similar conclusions with respect to (1) identification of experimental outliers, (2) overall assessment of robustness and inter-laboratory reproducibility and (3) re-classification of the unknown compounds to the respective toxicity classes. In summary, the developed bioinformatics workflows deliver accurate measures for inter-laboratory comparison studies, and the study can be used as guidance for validation of future carcinogenicity assays in order to implement testing of human in vitro alternatives to animal testing.
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Carcinógenos/toxicidad , Biología Computacional , Perfilación de la Expresión Génica , Túbulos Renales Proximales/efectos de los fármacos , Ensayos de Aptitud de Laboratorios , Hígado/efectos de los fármacos , Toxicogenética/métodos , Transcriptoma/efectos de los fármacos , Carcinógenos/clasificación , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Estudio de Asociación del Genoma Completo , Humanos , Túbulos Renales Proximales/metabolismo , Hígado/metabolismo , Variaciones Dependientes del Observador , Análisis de Secuencia por Matrices de Oligonucleótidos , Reproducibilidad de los Resultados , Medición de Riesgo , Factores de Tiempo , Flujo de TrabajoRESUMEN
Efforts are put into developing toxicogenomics-based toxicity testing methods using in vitro human cell models for improving human risk assessment/replacing animal models. Human in vitro liver models include HepG2, HepaRG and primary human hepatocytes (PHH). Studies on comparability/applicability of these cell types mainly focus on assessing baseline biotransformation capacities/cytochrome P450-inducibility, but compound-induced gene expression profiles are at least as important. Therefore, we compared baseline and aflatoxin B1- and benzo(α)pyrene-induced gene expression profiles in HepG2, HepaRG and PHH (11-13 donors). At baseline, all liver models differ from each other with respect to whole genome gene expression levels. PHH show profound inter-individual differences, and are most similar to HepaRG. After compound exposure, induced gene expression profiles are more similar between cell models, especially for benzo(α)pyrene. Pathways involved in compound metabolism are induced in all 3 models, while others are more pronounced in a specific cell model. Examples are transcriptomic modifications of carbohydrate-related genes (HepaRG) and of receptor-related genes (PHH) after benzo(α)pyrene exposure, and of cell cycle-related genes (HepG2) after aflatoxin B1 exposure. PHH gene expression responses are the most heterogeneous. In conclusion, at base line level PHH are more similar to HepaRG than to HepG2, but for toxicogenomics applications both cell lines perform equally well in comparison to PHH.
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Alternativas al Uso de Animales/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Mutágenos/farmacología , Toxicogenética/métodos , Adulto , Aflatoxina B1/farmacología , Aflatoxina B1/toxicidad , Benzo(a)pireno/farmacología , Benzo(a)pireno/toxicidad , Sistema Biliar/citología , Sistema Biliar/efectos de los fármacos , Sistema Biliar/metabolismo , Biotransformación , Línea Celular Tumoral , Células Cultivadas , Niño , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Femenino , Perfilación de la Expresión Génica , Células Hep G2 , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Lactante , Masculino , Mutágenos/toxicidad , Análisis de Componente Principal , Reproducibilidad de los ResultadosRESUMEN
Synopsis Octadecenedioic acid is known as a skin whitening agent but its activity is not mediated via a direct inhibition of tyrosinase. Based on the secondary properties of this molecule, such as its anti-inflammatory and anti-ageing effects, we postulated that octadecenedioic acid interacted with the peroxisome proliferator-activated receptor (PPAR) as this nuclear receptor also mediates these effects. Using reporter gene technology, we were indeed able to demonstrate binding of octadecenedioic acid to all three PPAR subtypes, in particular PPARgamma with an EC(50)-value of approx. 1 x 10(-6) m. Binding to PPARgamma of octadecenedioic acid or rosiglitazone, a known pharmaceutical PPARgamma agonist, led to reduced melanogenesis. Subsequently also tyrosinase mRNA (as measured by real-time polymerase chain reaction) and tyrosinase levels (as measured by Western blot) were reduced, suggesting the existence of a complete novel mechanism of skin whitening agents: binding to PPARgamma results in reduced tyrosinase mRNA expression which in turn results in less tyrosinase being formed. This in turn leads to reduced melanogenesis both in vitro and in vivo Because octadecenedioic acid binds not only to PPARgamma but also to PPARalpha and PPARdelta, other efficacies mediated via these receptors may also be expected.
RESUMEN
Most of the slimming products already developed for cosmetic applications did not result from strategies that integrate whole lipolysis-regulating mechanisms. We thus focused our attention on a more complete integration of these mechanisms and we developed slimming liposomes (SLC) containing two micro-circulation activators, i.e., esculoside and Centella asiatica extracts, one phosphodiesterase inhibitor, i.e., caffeine, and one fatty acid-beta oxidation activator, i.e., L-carnitine. The validity of our approach was assessed through (a) in vitro tests demonstrating that SLC induced a dramatic increase in the cyclic adenosine monophosphate (cAMP) content in human adipocytes, with a subsequent rise in the nonesterified fatty acids (NEFA) content of human adipocyte incubation medium, and (b) in vivo studies showing that SLC could provide an actual potent slimming effect on human volunteers. Moreover, we give here, through binding experiments, the unambiguous demonstration that SLC is able to antagonize the alpha(2)-adrenergic receptor that is known to reduce intracellular AMPc content and, subsequently, to down-regulate lipolysis. This alpha(2)-adrenergic antagonism has never been reported for any component of SLC, and this work is the first demonstration of the alpha(2)-adrenergic antagonism of such a combination of active liposome compounds.
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Antagonistas de Receptores Adrenérgicos alfa 2 , Lipólisis , Liposomas , Pérdida de Peso/efectos de los fármacos , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Antagonistas Adrenérgicos alfa/farmacología , AMP Cíclico/metabolismo , Técnicas In VitroAsunto(s)
Alternativas a las Pruebas en Animales , Absorción Intestinal/fisiología , Mucosa Intestinal/fisiología , Animales , Células CACO-2 , Directrices para la Planificación en Salud , Humanos , Mucosa Intestinal/enzimología , Modelos Biológicos , Técnicas de Cultivo de Órganos , Xenobióticos/farmacologíaRESUMEN
A prevalidation study on in vitro tests for acute skin irritation was conducted during 1999 and 2000. The overall objective of validation in this area, of which this prevalidation study is an initial stage, is to identify tests capable of discriminating irritants (I) from non-irritants (NI), as defined according to European Union (EU) risk phrases ("R38"; no classification) and the harmonised OECD criteria ("Irritant"; no label). This prevalidation study specifically addressed aspects of: protocol refinement (phase I), protocol transfer (phase II), and protocol performance (phase III), in accordance with the prevalidation scheme defined by the European Centre for the Validation of Alternative Methods (ECVAM). The tests evaluated were: EpiDerm (phases I, II and III), EPISKIN (phases I, II and III), PREDISKIN (phases I and II, and additional protocol refinement), the non-perfused pig ear method (phases I and II, and additional protocol refinement), and the mouse skin integrity function test (SIFT; phases I and II). Modified, standardised test protocols and well-defined prediction models were available for each of the tests at the end of phase I. The results of phase I (intralaboratory reproducibility) were sufficiently promising for all of the tests to progress to phase II. Protocol transfer between the Lead Laboratory and Laboratory 2 was undertaken for all five tests during phase II, and additional refinements were made to the test protocols. For EpiDerm, EPISKIN and the SIFT, the intralaboratory and interlaboratory reproducibilities were acceptable; however, better standardisation of certain aspects of the test protocols was needed prior to commencing phase III. Neither PREDISKIN nor the pig ear test performed sufficiently well in phase II to progress to phase III. The PREDISKIN protocol was overly sensitive, resulting in the prediction of all the NI chemicals as I. The variability in the pig ear test results was too great, indicating that the test would show limited predictive ability. In additional studies (a repeat of phase I), further modification of the PREDISKIN protocol and a change in the prediction model considerably improved the ability of the test to distinguish I from NI chemicals. However, attempts to improve the intralaboratory reproducibility of the pig ear test were unsuccessful. In phase III an initial assessment of the reproducibility and predictive ability, in three independent laboratories per test, was undertaken for the EpiDerm and EPISKIN tests (the SIFT was a late inclusion in the prevalidation study, and is being evaluated in a separate phase III study). A set of 20 coded chemicals (10 I, 10 NI) were tested with the final, refined, test protocols. The intralaboratory reproducibility was acceptable for both EpiDerm and EPISKIN. The interlaboratory reproducibility was considered to be acceptable for EPISKIN; however, for EpiDerm, analysis of variance (ANOVA) indicated that there was a statistically significant laboratory effect on the overall variability, suggesting that the interlaboratory transferability of the test needs to be improved. The EpiDerm test had an overall accuracy of 58%, with an over-prediction rate of 37% and an under-prediction rate of 47%. The EPISKIN test had an overall accuracy of 58%, showing an under-prediction rate of 23% and an over-prediction rate of 60%. It is concluded that, as yet, none of the tests evaluated in this prevalidation study are ready for inclusion in a formal validation study on in vitro tests for acute skin irritation. Overall protocol performance of the SIFT is currently being evaluated in a phase III study. Further studies are also in progress to improve the test protocols and prediction models for EpiDerm and EPISKIN.
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Alternativas a las Pruebas en Animales , Dermatitis por Contacto/inmunología , Irritantes/efectos adversos , Pruebas de Irritación de la Piel , Piel/inmunología , Animales , Técnicas de Cultivo de Célula , Oído , Epidermis/efectos de los fármacos , Epidermis/ultraestructura , Humanos , Irritantes/inmunología , Ratones , Reproducibilidad de los Resultados , Proyectos de Investigación , Piel/citología , Piel/efectos de los fármacos , PorcinosRESUMEN
In the present study we evaluated viability and detoxifying enzyme capacity of cryopreserved hepatocytes from various species, including man, immobilized in calcium alginate gels. Ethoxyresorufin O-deethylase, phenacetin deethylase, pentoxyresorufin O-dealkylase, tolbutamide hydroxylase, S-mephenytoin hydroxylase, dextromethorphan demethylase, and nifedipine oxidation corresponding to the major cytochromes P450 (CYP) involved in xenobiotic metabolism as well as whole glutathione S-transferase (GST) activity were measured using specific substrates and after exposure or not to prototypical inducers. After deep-freeze storage, viability of immobilized hepatocytes was only slightly reduced and most CYP-related monooxygenase activities were well preserved, being expressed at levels close to those measured in unfrozen hepatocyte monolayers. By contrast, total GST activity was decreased by around 50%. However, as did CYP1A- and 3A-related enzymes, rat GST remained capable of responding to prototypical inducers. The fold increases were comparable in unfrozen and frozen immobilized hepatocytes and in unfrozen hepatocyte monolayers. The duration of storage, even when exceeding one year, did not affect viability and functions. In conclusion, after cryopreservation, alginate-entrapped hepatocytes remain highly viable and capable of expressing most detoxifying enzymes at levels close to those expressed in corresponding unfrozen hepatocyte monolayers and of responding to prototypical inducers.
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Células Inmovilizadas/citología , Células Inmovilizadas/enzimología , Hepatocitos/citología , Hepatocitos/enzimología , Inactivación Metabólica/fisiología , Alginatos , Animales , Supervivencia Celular , Criopreservación , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP2B1/metabolismo , Perros , Geles , Glutatión Transferasa/metabolismo , Haplorrinos , Humanos , Técnicas In Vitro , RatasRESUMEN
Primary cultures of Pecten maximus heart cells, isolated by an enzymatic procedure, were routinely obtained with a high level of reproducibility in a simple medium based on sterile seawater. Cells attached to the plastic substratum without the need to add a special factor. The number of adhering cells gradually increased with the time of culture. Two types of adhering cells were observed: epitheliallike cells and fibroblastlike cells, which were more numerous. The latter cells were identified as myocytes by electron microscopy and immunofluorescent staining. Results obtained by autoradiography, after incorporation of [14C]leucine, [3H]thymidine, and [14C]acetate, confirmed functional activity of the cells. These cultures were maintained viable in vitro during at least 1 mo.
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Moluscos/citología , Animales , Autorradiografía , Adhesión Celular , Diferenciación Celular , División Celular , Separación Celular , Células Cultivadas , Medios de Cultivo , ADN/biosíntesis , Células Epiteliales/citología , Fibroblastos/citología , Técnica del Anticuerpo Fluorescente , Microscopía Electrónica , Miocardio/citología , Agua de Mar , Factores de TiempoRESUMEN
Ethical, economic and technical reasons hinder regular supply of freshly isolated hepatocytes from higher mammals such as monkey for preclinical evaluation of drugs. Hence, we aimed at developing optimal and reproducible protocols to cryopreserve and thaw parenchymal liver cells from this major toxicological species. Before the routine use of these protocols, we validated them through a multi-laboratory study. Dissociation of the whole animal liver resulted in obtaining 1-5 billion parenchymal cells with a viability of about 86%. An appropriate fraction (around 20%) of the freshly isolated cells was immediately set in primary culture and various hepato-specific tests were performed to examine their metabolic, biochemical and toxicological functions as well as their ultrastructural characteristics. The major part of the hepatocytes was frozen and their functionality checked using the same parameters after thawing. The characterization of fresh and thawed monkey hepatocytes demonstrated the maintenance of various hepato-specific functions. Indeed, cryopreserved hepatocytes were able to survive and to function in culture as well as their fresh counterparts. The ability for synthesis (proteins, ATP, GSH) and conjugation and secretion of biliary acids was preserved after deep freeze storage. A better stability of drug metabolizing activities than in rodent hepatocytes was observed in monkey. After thawing, Phase I and Phase II activities (cytochrome P450, ethoxycoumarin-O-deethylase, aldrin epoxidase, epoxide hydrolase, glutathione transferase, glutathione reductase and glutathione peroxidase) were well preserved. The metabolic patterns of several drugs were qualitatively and quantitatively similar before and after cryopreservation. Lastly, cytotoxicity tests suggested that the freezing/thawing steps did not change cell sensitivity to toxic compounds.
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Criopreservación , Evaluación Preclínica de Medicamentos/métodos , Hígado/fisiología , Preservación de Órganos , Adenosina Trifosfato/metabolismo , Amodiaquina/toxicidad , Animales , Supervivencia Celular , Células Cultivadas , Enzimas/metabolismo , Eritromicina/toxicidad , Estudios de Evaluación como Asunto , Furosemida/toxicidad , Glutatión/metabolismo , Hígado/citología , Hígado/efectos de los fármacos , Macaca fascicularis , Masculino , Pruebas de ToxicidadRESUMEN
1. The metabolism of Meloxicam (ME) and the cytochrome(s) P450 (CYPs) involved were analysed by using primary human hepatocytes, human liver microsomes and microsomes from recombinant human B-lymphoblastoid cell lines. 2. While human hepatocytes were capable of converting ME to a 5-hydroxymethyl metabolite (M7) and then to a 5-carboxyderivative (M5), human liver microsomes formed mostly only the 5-hydroxymethylderivative. The kinetics of the formation of M7 by human liver microsomes were biphasic with Km = 13.6 +/- 9.5 and 381 +/- 55.2 microM respectively. The corresponding Vmax were 33.7 +/- 24.2 and 143 +/- 83.9 pmol/min/mg protein respectively. 3. CYP2C9 and, to a much lesser extent, CYP3A4 were found to convert ME to M7. The involvement of 2C9 was demonstrated by inhibition of tolbutamide hydroxylase activity in the presence of ME, inhibition of ME metabolism by sulphaphenazole, correlation between ME metabolism and tolbutamide hydroxylase activity and active metabolism of ME by recombinant 2C9. The involvement of 3A4 was shown by inhibition of ME metabolism by ketoconazole, correlation between ME metabolism and nifedipine oxidase activity and metabolism of ME by recombinant 3A4. Kinetics of the formation of M7 by the individual enzymes resulted in a Km = 9.6 microM and Vmax = 8.4 pmol/min/mg protein for 2C9 and a Km = 475 microM and Vmax = 23 pmol/min/mg protein for 3A4.
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Antiinflamatorios no Esteroideos/metabolismo , Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/metabolismo , Hígado/enzimología , Oxigenasas de Función Mixta/metabolismo , Esteroide 16-alfa-Hidroxilasa , Esteroide Hidroxilasas/metabolismo , Tiazinas/metabolismo , Tiazoles/metabolismo , Citocromo P-450 CYP2C9 , Citocromo P-450 CYP3A , Inhibidores Enzimáticos del Citocromo P-450 , Inhibidores Enzimáticos/farmacología , Humanos , Hidroxilación , Cinética , Hígado/citología , Meloxicam , Microsomas Hepáticos/enzimología , Oxigenasas de Función Mixta/antagonistas & inhibidores , Esteroide Hidroxilasas/antagonistas & inhibidores , Especificidad por Sustrato , Células Tumorales CultivadasRESUMEN
Rat hepatocytes immobilized in calcium-alginate beads were tested for their ability to survive and function in vitro by comparison with hepatocyte monolayer cultures before and after cryopreservation. The freeze-thaw protocol previously designed for suspended hepatocytes was used; the freezing medium was the Leibovitz medium containing 10% fetal calf serum and 16% dimethylsulfoxide. The functions investigated included protein, lipid, and urea synthesis, albumin secretion, glycogen content, and various phase I and phase II enzyme activities. Cell viability was not altered by immobilization and the freeze-thaw procedure. Most functions tested were expressed at similar levels in unfrozen immobilized hepatocytes and corresponding hepatocyte monolayers. Only protein neosynthesis and some phase II drug metabolizing enzyme activities were decreased. The freeze-thaw process had no detrimental effect, whatever the function investigated. In addition, cryopreserved immobilized hepatocytes remained responsive to inducers: ethoxyresorufin O-deethylase activity was increased 11-12 times by 3-methylcholanthrene treatment in both fresh and cryopreserved alginate-entrapped hepatocytes. These results clearly show that hepatocytes immobilized in calcium-alginate beads remain functional even after cryopreservation indicating that they represent a promising approach for xenobiotic metabolism and toxicity studies.
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Alginatos/farmacología , Criopreservación , Hígado/citología , Xenobióticos/metabolismo , Animales , Adhesión Celular , Supervivencia Celular , Ácido Glucurónico , Ácidos Hexurónicos , Hígado/metabolismo , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Oxadiazon [5-ter-butyl-3-(2,4-dichloro-5-isopropoxyphenyl)- 1,3,4-oxadiazol-2(3H)-one] was administered orally at 20-500 mg/ kg body wt/day to male Sprague-Dawley CD rats for 14 days, at 20-200 mg/kg body wt/day to male CD1 [CR1/CD-1(1GR)BR] mice for 28 days, and at 500 mg/kg body wt/day to male beagle dogs for 28 days. Although liver enlargement was observed in the three species, morphological studies indicated that peroxisome proliferation only occurred in rats and mice. Parallel biochemical investigations showed that there was a dose-dependent increase in the peroxisomal cyanide-insensitive palmitoyl CoA oxidase and acetyl carnitine transferase activities in treated rats and mice. Acetyl carnitine activity appeared to correlate well with the number and volume of peroxisomes as determined histologically. The increases in enzyme activities at 200 mg/kg body wt/day oxadiazon were comparable in rats and mice indicating that both rodent species were equally sensitive to oxadiazon-induced peroxisome proliferation. When added in vitro to cultured rat hepatocytes at concentrations ranging from 2.5 to 10 x 10(-5) M, oxadiazon induced a dose-dependent increase in the activities of palmitoyl CoA oxidase and acetyl carnitine transferase. The ratio obtained by comparing oxadiazon and clofibric acid on acetyl carnitine transferase activity at 5 x 10(-5) M in the present in vitro study on rat hepatocytes are equivalent to those that can be calculated from the results on this enzyme activity obtained in vivo in the rat with 500 mg/ kg body wt/day oxadiazon (this study) and clofibric acid (literature values), indicating that the rat hepatocyte cultures gave satisfactory results regarding peroxisome proliferation. Neither oxadiazon nor clofibric acid modified the activities of palmitoyl CoA oxidase and acetyl carnitine transferase in cultured human hepatocytes. The results presented here demonstrate clearly that oxadiazon induces peroxisome proliferation in rodents in vivo and in vitro, as determined both biochemically and morphologically, whereas dogs in vivo and human hepatocytes in vitro were refractory to peroxisome proliferation. This observation extends to the herbicide oxadiazon, which is structurally unrelated to other known peroxisome proliferators, the generally observed marked species difference in sensitivity to peroxisome proliferation, and has important implications in the human safety evaluation of this herbicide.
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Herbicidas/farmacología , Hígado/efectos de los fármacos , Microcuerpos/metabolismo , Oxadiazoles/farmacología , Animales , Peso Corporal/efectos de los fármacos , Células Cultivadas , Perros , Humanos , Hígado/enzimología , Hígado/metabolismo , Hígado/ultraestructura , Masculino , Ratones , Tamaño de los Órganos/efectos de los fármacos , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Especificidad de la EspecieRESUMEN
The aim of this study was to analyse viability, growth, differentiation and drug metabolic capacity of cultured human keratinocytes obtained from post-mortem skin. Epidermal cells were prepared from 1-day post-mortem paired sun-exposed (outer) and sun-protected (inner) sites of the upper arm, of donors aged 47-80 years. The percentage of viable cells obtained from post-mortem skin was only slightly lower than that usually obtained for keratinocytes isolated from fresh skin, and no alterations of epidermal markers were noted. Keratinocytes isolated post-mortem from non-exposed skin had a higher viability (78 versus 73%), and a more active proliferation, while their attachment rate, keratin composition, lipid synthesis capacity and transglutaminase activity levels were similar to those of epidermal cells obtained from the sun-exposed skin. Keratinocytes isolated from post-mortem skin expressed various phase I and II activities at levels similar to those obtained with keratinocytes isolated from fresh skin while drug metabolizing enzyme activities were consistently higher in sun-exposed compared to sun-protected cells. The results support the conclusion that skin collected post-mortem can represent an alternative source of viable and functional epidermal cells, and that the functional changes that occur in adult keratinocytes habitually exposed to the sun, affect much more strongly the drug metabolism capacity than the expression of differentiation markers.
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Queratinocitos/metabolismo , Queratinocitos/patología , Cambios Post Mortem , Piel/metabolismo , Piel/patología , Anciano , Anciano de 80 o más Años , Biotransformación , Adhesión Celular , Técnicas de Cultivo de Célula , División Celular , Supervivencia Celular , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Queratinas/análisis , Lípidos/biosíntesis , Masculino , Persona de Mediana Edad , Piel/efectos de la radiación , Luz Solar , Transglutaminasas/metabolismoRESUMEN
In this study we analysed the expression and induction of several drug metabolizing enzymes involved in either phase I or phase II reactions, in adult human keratinocytes cultured in submerged conditions. We also evaluated the influence of confluence, subcultivation and cryopreservation on the expression of these enzymes. Besides ethoxyresorufin-O-deethylase (EROD) and glutathione S-transferase (GST) activities, which have been shown previously to be maintained in such cultures, three additional enzyme activities were measured (i.e. phenacetin deethylase, a phase I enzyme, and procainamide N-acetyltransferase and paracetamol sulfotransferase, two phase II enzymes). Post-confluent keratinocytes showed decreased activities in comparison with preconfluent cells and the different enzymes tested revealed different patterns. After confluence, some activities, such as those of procainamide N-acetyltrans-ferase, phenacetin deethylase and paracetamol sulfotransferase, showed only a slight decrease, whereas EROD and GST activities were decreased by 65 and 50%, respectively. No major differences were observed between keratinocytes in primary culture and those in second subculture. After freezing, xenobiotic metabolizing enzyme activities were only slightly reduced, if at all. Induction of EROD and GST enzymes was also analysed. Maximum EROD activity was obtained with 1 muM 3-methylcholanthrene (3-MC) and 20 muM benzanthracene (BA), in both pre-confluent and post-confluent cultures. At their optimal concentration 3-MC was a stronger inducer than BA. GST activity was slightly induced by the different compounds tested only in pre-confluent keratinocytes. In conclusion, the presence of a variety of drug metabolizing enzymes in adult human keratinocytes cultured in submerged conditions suggests that this model is suitable for investigating epidermal biotransformation of drugs and other chemicals and for determining the potential cutaneous toxicity of metabolites.
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To determine whether compounds are able to reach the neural microenvironment, a blood-brain barrier (BBB) co-culture model has been recently developed with bovine brain capillary endothelial cells and newborn rat astrocytes. In this study, the permeability of confluent endothelial cells to various compounds and the functional activity of P-glycoprotein (P-gp), an ATP-dependent pump known to efflux drugs from multidrug-resistant tumoral cells, was assessed. The permeability of the lipophilic compounds imipramine and sulpiride differed in relation to their structure. A good correlation was observed with in vivo brain extraction levels. P-gp activity was estimated by measuring the uptake of [(3)H]vinblastine by the endothelial cells, with or without verapamil, which is known to reverse drug resistance. Intracellular accumulation of the vinca alkaloid was strongly increased after addition of verapamil, suggesting that P-gp is active in these cells. These results provide further support for the use of the co-culture model of bovine brain endothelial cells and rat astrocytes to screen new centrally active drugs.
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We have studied the cytotoxity to human keratinocytes of three main classes of drugs known to induce cutaneous adverse reactions, namely beta-lactam antibiotics, non-steroidal anti-inflammatory drugs (NSAIDs) and sulfonamides, using the neutral red uptake as an endpoint. IC(50) values were determined for 21 drugs, after 20 hr of exposure and compared with those obtained with rat hepatocytes. NSAIDs were found to be more cytotoxic than beta-lactum antibiotics to human keratinocytes. Large variations in IC(50) values were obtained between molecules of a same class, as well as between keratinocyte cultures from different donors, especially for beta-lactum antibiotics. All NSAIDs and beta-lactam antibiotics tested were more cytotoxic to rat hepatocytes (1.6- to 27-fold). Both cell types were only slightly sensitive to sulfonamides, if at all.
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A cell kit named PREDISAFE based on the use of confluent rabbit fibroblastic cells has been designed to predict eye irritancy of cosmetic raw materials and formulations. The kit can be stored for a few days and/or shipped at room temperature. Cytotoxicity was estimated after 1 min or 15 min contact with test compounds using the neutral red release assay. For the 84 products tested, IC50 values gave intervals similar to classes defined from the Draize test, i.e., mild, moderate, severe and extreme irritancy.
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Supervivencia Celular/efectos de los fármacos , Cosméticos/toxicidad , Ojo/efectos de los fármacos , Irritantes/toxicidad , Juego de Reactivos para Diagnóstico , Pruebas de Toxicidad/métodos , Animales , Línea Celular , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Rojo Neutro/metabolismo , ConejosRESUMEN
We examined the effects of inflammatory cytokines (interleukin-1 beta, tumor necrosis factor-alpha and transforming growth factor-beta) on the plasminogen activator system (urokinase, tissue-type plasminogen activator, type 1 plasminogen activator inhibitor) in primary cultures of human hepatocytes. We show that interleukin-1 beta and tumor necrosis factor-alpha increase urokinase-type plasminogen activator production, reinforcing the concept that increased urokinase production is associated with inflammatory processes. By contrast, the same agents (i.e., interleukin-1 beta and tumor necrosis factor-alpha) do not stimulate plasminogen activator inhibitor type 1 production. This latter observation rules out hepatocytes as a major cellular source of plasmatic plasminogen activator inhibitor type 1 during acute-phase-related responses. Among the inflammatory agents used, transforming growth factor-beta was found to be the most effective modulator of both urokinase-type plasminogen activator and plasminogen activator inhibitor type 1, inducing severalfold increases of activity of urokinase-type plasminogen activator, antigen and the corresponding mRNA and increasing plasminogen activator inhibitor type 1 antigen and mRNA levels. Urokinase-type plasminogen activator and plasminogen activator inhibitor type 1 modulation by transforming growth factor-beta may play a critical role in hepatic pathophysiology.