RESUMEN
In vitro experiments have a high potential to improve current chemical safety assessment and reduce the number of animals used. However, most studies conduct hazard assessment alone, largely ignoring exposure and kinetic parameters. Therefore, in this study the kinetics of cyclosporine A (CsA) and the dynamics of CsA-induced cyclophilin B (Cyp-B) secretion were investigated in three widely used hepatic in vitro models: primary rat hepatocytes (PRH), primary human hepatocytes (PHH) and HepaRG cells. Cells were exposed daily to CsA for up to 14 days. CsA in cells and culture media was quantified by LC-MS/MS and used for pharmacokinetic modeling. Cyp-B was quantified by western blot analysis in cells and media. All cell systems took up CsA rapidly from the medium after initial exposure and all showed a time- and concentration-dependent Cyp-B cellular depletion and extracellular secretion. Only in PRH an accumulation of CsA over 14 days repeated exposure was observed. Donor-specific effects in CsA clearance were observed in the PHH model and both PHH and HepaRG cells significantly metabolized CsA, with no bioaccumulation being observed after repeated exposure. The developed kinetic models are described in detail and show that all models under-predict the in vivo hepatic clearance of CsA, but to different extents with 27-, 24- and 2-fold for PRH, PHH and HepaRG cells, respectively. This study highlights the need for more attention to kinetics in in vitro studies.
Asunto(s)
Ciclosporina/farmacocinética , Hepatocitos/metabolismo , Adulto , Anciano , Animales , Células Cultivadas , Humanos , Masculino , Persona de Mediana Edad , RatasRESUMEN
The assumption that compounds with similar toxic endpoints generate unique gene expression signatures has led to attempts to classify unknown compounds according to their genomic profile. However, studies reported so far have mostly been conducted in vivo. In this proof of concept study, we assessed the use of rat hepatocyte sandwich cultures in combination with a toxicogenomic classification method to generate a predictive in vitro toxicity classification model. After pre-incubation for 3 days, primary rat hepatocytes were treated for up to 9 days with 13 well known model compounds, changes in the global gene expression profile were measured and subsequently used for the establishment of a predictive classification model. A subset of 724 genes was capable of discriminating compounds with a misclassification rate of 7.5%. As a preliminary verification, the resulting classifier was applied to two blinded control compounds. The classification of compounds according to transient changes in global gene expression allowed the correct prediction independently from the knowledge of their underlying toxic mechanisms. The results of this first pilot study demonstrate the possibility of in vitro gene expression models to contribute to candidate selection early in drug discovery by improving the predictivity of toxicological studies and thereby reducing animal usage in toxicology.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Drogas en Investigación/toxicidad , Regulación de la Expresión Génica/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Modelos Biológicos , Pruebas de Toxicidad/métodos , Alternativas al Uso de Animales , Animales , Técnicas de Cultivo de Célula , Células Cultivadas , Colágeno Tipo I/metabolismo , Drogas en Investigación/clasificación , Estudios de Factibilidad , Perfilación de la Expresión Génica , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Concentración Osmolar , Proyectos Piloto , Ratas , Ratas Wistar , Toxicogenética/métodosRESUMEN
It is important to investigate the induction of cytochrome P450 (CYP) enzymes by drugs. The most relevant end point is enzyme activity; however, this requires many cells and is low throughput. We have compared the CYP1A, CYP2B and CYP3A induction response to eight inducers in rat and human hepatocytes using enzyme activities (CYP1A2 (ethoxyresorufin), 2B (benzoxyresorufin for rat and bupropion for human) and CYP3A (testosterone)) and Taqman Low Density Array (TLDA) analysis. There was a good correlation between the induction of CYP1A2, CYP2B6 and CYP3A4 enzyme activities and mRNA expression in human hepatocytes. In contrast, BROD activities and mRNA expression in rat hepatocytes correlated poorly. However, bupropion hydroxylation correlated well with Cyp2b1 expression in rat hepatocytes. TLDA analysis of a panel of mRNAs encoding for CYPs, phase 2 enzymes, nuclear receptors and transporters revealed that the main genes induced by the 8 compounds tested were the CYPs. AhR ligands also induced UDP-glucuronosyltransferases and glutathione S-transferases in rat and human hepatocytes. The transporters, MDR1, MDR3 and OATPA were the only transporter genes significantly up-regulated in human hepatocytes. In rat hepatocytes Bsep, Mdr2, Mrp2, Mrp3 and Oatp2 were up-regulated. We could then show a good in vivo:in vitro correlation in the induction response of isolated rat hepatocytes and ex-vivo hepatic microsomes for the drug development candidate, EMD392949. In conclusion, application of TLDA methodology to investigate the potential of compounds to induce enzymes in rat and human hepatocytes increases the throughput and information gained from one assay, without reducing the predictive capacity.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Inducción Enzimática/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , ARN Mensajero/metabolismo , Anciano , Animales , Células Cultivadas , Sistema Enzimático del Citocromo P-450/genética , Humanos , Masculino , ARN Mensajero/genética , Ratas , Ratas Wistar , Regulación hacia ArribaRESUMEN
The surprising findings that male mice lacking the estrogen receptor (ER) alpha (alphaERKO) have atrophic testis and are infertile proved that ERalpha is involved in normal testicular function. To obtain compatible in vitro model systems for alphaERKO male mice, we immortalized different cell lines from the testis of alphaERKO and wild-type (C57BL/6) mice with the human papilloma virus E6/E7 genes. The established cell lines were characterized for Sertoli, Leydig, and peritubular cell markers by means of messenger RNA expression and functional assays. One wild-type-derived cell line showed Leydig cell-specific marker gene expression and produced testosterone after stimulation with cyclic adenosine monophosphate. Most wild-type cell lines expressed androgen receptor and a functional ERalpha as shown by high estrogenic activity in a luciferase-based transactivation assay. Most notably, the wild-type-derived WL3, and the ES4 cell line derived from alphaERKO mice expressed ERbeta and showed ER-mediated transcriptional activity, but no ERalpha protein expression. These cell lines with and without functional ERalpha or ERbeta enable the analyses of ER subtype-specific responses and their function in testicular cell signaling, morphogenesis, and neoplasia.
Asunto(s)
Células Intersticiales del Testículo/citología , Receptores de Estrógenos/genética , Células de Sertoli/citología , Factores de Edad , Animales , Línea Celular Transformada , Receptor alfa de Estrógeno , Receptor beta de Estrógeno , Expresión Génica/fisiología , Técnicas In Vitro , Células Intersticiales del Testículo/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/análisis , Células de Sertoli/fisiología , Maduración SexualRESUMEN
The estrogen receptors ERalpha and ERbeta are the main mediators of estrogen action and estrogens play an important role in a variety of aspects of physiology besides their well acknowledged function in reproduction. In vivo and in vitro studies indicate that the estrogen receptors are mechanistically implicated in endocrine-related diseases. Recent studies with estrogen receptor knockout mice have helped to unravel the role of the estrogen receptors in brain degeneration, osteoporosis, cardiovascular diseases and obesity.
Asunto(s)
Enfermedades del Sistema Endocrino/etiología , Receptores de Estrógenos/fisiología , Tejido Adiposo/metabolismo , Animales , Huesos/metabolismo , Encéfalo/metabolismo , Enfermedades Cardiovasculares/etiología , Enfermedades Cardiovasculares/metabolismo , Sistema Cardiovascular/metabolismo , Enfermedades del Sistema Endocrino/metabolismo , Receptor alfa de Estrógeno , Receptor beta de Estrógeno , Ratones , Ratones Noqueados , Enfermedades Neurodegenerativas/etiología , Enfermedades Neurodegenerativas/metabolismo , Obesidad/etiología , Obesidad/metabolismo , Osteoporosis/etiología , Osteoporosis/metabolismo , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismoRESUMEN
While testing for genotoxicity is usually performed on single chemicals, exposure of humans always comprises a number of genotoxic agents. The investigation of potentially synergistic effects of combinations therefore is an important issue in toxicology. Combinations of 511 keV gamma-radiation with the chemical alkylating agent ethyl methane-sulfonate were investigated in the in vitro micronucleus test in mouse lymphoma L5178Y cells. With combinations in the low dose linear effect range for the individual agents (0. 25-2 Gy and 0.8-3.2 mM, respectively), supra-additivity by 34-86% was seen. The synergism was more pronounced at the higher dose levels. Supra-additivity was confirmed in experiments using cytochalasin B and analyzing binucleate cells only, to control for putative effects on the cell cycle. Statistical significance was shown by a 2-factor analysis of variance with interaction. The results indicate that damage to DNA by gamma-radiation and alkylation could affect different rate limiting steps in the formation of micronuclei. Further investigations will have to show whether the observations are of general validity, in particular, whether other end-points of genotoxicity produce the same results and whether the degree of supra-additivity is always dose dependent. The latter would have a strong impact on risk assessment for mixtures at low doses.
Asunto(s)
Metanosulfonato de Etilo/toxicidad , Rayos gamma , Mutágenos/toxicidad , Animales , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Leucemia L5178 , Ratones , Micronúcleos con Defecto Cromosómico/efectos de los fármacos , Micronúcleos con Defecto Cromosómico/efectos de la radiación , Pruebas de Micronúcleos , Células Tumorales CultivadasRESUMEN
A close association between Helicobacter pylori infection and the development of gastric adenocarcinoma in humans has been demonstrated. Therefore, the direct induction of DNA damage by H. pylori was investigated here using the in vitro micronucleus assay. After 5 days of incubation with bacterial lysate a dose-dependent formation of micronuclei was found, which was not limited to cytotoxic protein concentrations and was not observed after treatment with Escherichia coli lysate (control). This induction of DNA damage may be a link between chronic H. pylori infection and development of adenocarcinoma of the stomach.
Asunto(s)
Daño del ADN , Helicobacter pylori , Pruebas de Micronúcleos , Animales , Supervivencia Celular , Escherichia coli , RatonesAsunto(s)
Glándulas Mamarias Animales/citología , ARN , Receptores de Estrógenos/fisiología , Animales , Línea Celular Transformada , Proteínas de Unión al ADN , Receptor alfa de Estrógeno , Femenino , Glándulas Mamarias Animales/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Estrógenos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Telomerasa/genéticaRESUMEN
1,8-Dihydroxyanthraquinones, present in laxatives, fungi imperfecti, Chinese herbs and possibly vegetables, are in debate as human carcinogens. We screened a variety of vegetables (cabbage lettuce, beans, peas), some herbs and herbal-flavoured liquors for their content of the 'free' anthraquinones emodin, chrysophanol and physcion. For qualitative and quantitative analysis, reversed-phase HPLC (RP-LC), gas chromatography-mass spectrometry (GC-MS) and RP-LC-MS were used. The vegetables showed a large batch-to-batch variability, from 0.04 to 3.6, 5.9 and 36 mg total anthraquinone per kg fresh weight in peas, cabbage lettuce, and beans, respectively. Physcion predominated in all vegetables. In the herbs grape vine leaves, couch grass root and plantain herb, anthraquinones were above the limit of detection. Contents ranged below 1 mg/kg (dry weight). All three anthraquinones were also found in seven of 11 herbal-flavoured liquors, in a range of 0.05 mg/kg to 7.6 mg/kg. The genotoxicity of the analysed anthraquinones was investigated in the comet assay, the micronucleus test and the mutation assay in mouse lymphoma L5178Y tk+/- cells. Emodin was genotoxic, whereas chrysophanol and physcion showed no effects. Complete vegetable extract on its own did not show any effect in the micronucleus test. A lettuce extract completely abolished the induction of micronuclei by the genotoxic anthraquinone danthron. Taking into consideration the measured concentrations of anthraquinones, estimated daily intakes, the genotoxic potency, as well as protective effects of the food matrix, the analysed constituents do not represent a high priority genotoxic risk in a balanced human diet.
Asunto(s)
Antraquinonas/toxicidad , Carcinógenos/toxicidad , Mutágenos/toxicidad , Plantas/efectos de los fármacos , Animales , Antraquinonas/análisis , Pruebas de Carcinogenicidad , Catárticos/análisis , Catárticos/toxicidad , Emodina/análogos & derivados , Emodina/análisis , Emodina/toxicidad , Humanos , Magnoliopsida/química , Ratones , Pruebas de Micronúcleos , Pruebas de Mutagenicidad , Extractos Vegetales/química , Plantas/genética , Células Tumorales Cultivadas , Verduras/químicaRESUMEN
Naturally occurring 1,8-dihydroxyanthraquinones are under consideration as possible carcinogens. Here we wanted to elucidate a possible mechanism of their genotoxicity. All three tested anthraquinones, emodin, aloe-emodin, and danthron, showed capabilities to inhibit the non-covalent binding of bisbenzimide Hoechst 33342 to isolated DNA and in mouse lymphoma L5178Y cells comparable to the topoisomerase II inhibitor and intercalator m-amsacrine. In a cell-free decatenation assay, emodin exerted a stronger, danthron a similar and aloe-emodin a weaker inhibition of topoisomerase II activity than m-amsacrine. Analysis of the chromosomal extent of DNA damage induced by these anthraquinones was performed in mouse lymphoma L5178Y cells. Anthraquinone-induced mutant cell clones showed similar chromosomal lesions when compared to the topoisomerase II inhibitors etoposide and m-amsacrine, but were different from mutants induced by the DNA alkylator ethyl methanesulfonate. These data support the idea that inhibition of the catalytic activity of topoisomerase II contributes to anthraquinone-induced genotoxicity and mutagenicity.
Asunto(s)
Antraquinonas/toxicidad , Inhibidores de Topoisomerasa II , Aloe/química , Animales , Bencimidazoles , Electroforesis en Gel de Agar , Emodina/análogos & derivados , Emodina/toxicidad , Colorantes Fluorescentes , Leucemia L5178 , Estructura Molecular , Pruebas de Mutagenicidad , Plantas Medicinales , Reacción en Cadena de la Polimerasa/métodos , Células Tumorales CultivadasRESUMEN
The studies presented here were designed to elucidate the enzymes involved in the biotransformation of naturally occurring 1, 8-dihydroxyanthraquinones and to investigate whether biotransformation of 1,8-dihydroxyanthraquinones may represent a bioactivation pathway. We first studied the metabolism of emodin (1, 3,8-trihydroxy-6-methylanthraquinone), a compound present in pharmaceutical preparations. With rat liver microsomes, the formation of two emodin metabolites, omega-hydroxyemodin and 2-hydroxyemodin, was observed. The rates of formation of omega-hydroxyemodin were not different with microsomes from rats that had been pretreated with inducers for different cytochrome P450 enzymes. Thus, the formation of omega-hydroxyemodin seems to be catalyzed by several cytochrome P450 enzymes at low rates. The formation of 2-hydroxyemodin was increased in liver microsomes from 3-methylcholanthrene-pretreated rats and was inhibited by alpha-naphthoflavone, by an anti-rat cytochrome P450 1A1/2 antibody, and, to a lesser degree, by an anti-rat cytochrome P450 1A1 antibody. These data suggest the involvement of cytochrome P450 1A2 in the formation of this metabolite. However, other cytochrome P450 enzymes also seem to catalyze this reaction. The anthraquinone chrysophanol (1,8-dihydroxy-3-methylanthraquinone) is transformed, in a cytochrome P450-dependent oxidation, to aloe-emodin (1, 8-dihydroxy-3-hydroxymethylanthraquinone) as the major product formed. The mutagenicity of the parent dihydroxyanthraquinones and their metabolites was compared in the in vitro micronucleus test in mouse lymphoma L5178Y cells. 2-Hydroxyemodin induced much higher micronucleus frequencies, compared with emodin. omega-Hydroxyemodin induced lower micronucleus frequencies, compared with emodin. Aloe-emodin induced significantly higher micronucleus frequencies than did chrysophanol. These data indicate that the cytochrome P450-dependent biotransformation of emodin and chrysophanol may represent bioactivation pathways for these compounds.
Asunto(s)
Antraquinonas/farmacocinética , Sistema Enzimático del Citocromo P-450/fisiología , Emodina/farmacocinética , Mutágenos/farmacocinética , Animales , Antraquinonas/toxicidad , Biotransformación , Emodina/toxicidad , Femenino , Hidroxilación , Cinética , Linfoma/genética , Masculino , Ratones , Micronúcleos con Defecto Cromosómico/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Ratas , Ratas WistarRESUMEN
We had reported that the plant-derived 1,8-dihydroxyanthraquinone derivatives, emodin and danthron, were clearly genotoxic in mouse lymphoma L5178Y cells, whereas chrysophanol was only weakly genotoxic and physcion not at all. Danthron was more potent than emodin. Furthermore, we had found that these compounds bound non-covalently to DNA and inhibited topoisomerase II activity. Interestingly, in these systems emodin was more potent than danthron. This inversion of the ranking prompted us to investigate the underlying mechanism. Since emodin shows a high serum-protein binding affinity, horse serum used as a media-supplement in the mouse lymphoma genotoxicity assays was analyzed for a potential selective scavenging of emodin. Non-covalent DNA-binding in mouse lymphoma L5178Y cells was investigated in the absence or presence of serum. In the presence of 10% serum, the DNA-binding potency of emodin was markedly reduced and was lower than that of danthron. We also applied mutation assays with mouse lymphoma cells and AS52 cells and varied the serum concentration used. In the absence of serum emodin showed slightly higher mutagenicity in AS52 cells than danthron. At reduced serum concentration (0.5%) emodin was strongly cytotoxic to the mouse lymphoma cells. For chrysophanol and physcion, a considerable reduction of the non-covalent DNA-binding potency in intact cells was found when compared to danthron, in concordance with their lower genotoxic potency. Overall, these data support the understanding that the genotoxicity of anthraquinones is, at least in part, mediated by non-covalent DNA-binding.
Asunto(s)
Antraquinonas/toxicidad , Mutágenos , Animales , Antraquinonas/química , ADN/metabolismo , Emodina/análogos & derivados , Emodina/toxicidad , Humanos , Leucemia L5178 , Inhibidores de Topoisomerasa II , Células Tumorales CultivadasRESUMEN
The enzymes tRNA guanine-transglycosylase (Tgt) and S-adenosylmethionine :tRNA ribosyltransferase-isomerase (QueA) participate in the biosynthesis of the hypermodified tRNA nucleoside queuosine (Q) in Escherichia coli. Here we show by HPLC analysis and gel retardation that both enzymes interact with an in vitro transcribed tRNA(ASP) from yeast, specifically modified with a Q precursor molecule. RNase I footprinting experiments showed strong protein tRNA contacts in the anticodon stem-loop and a minor interaction with the dihydrouridine loop. This suggests that all identity elements for the recognition of Q-specific tRNAs are clustered in the anticodon region and explains earlier results that both enzymes accept a RNA microhelix with the sequence of an anticodon stem-loop as substrate.