RESUMO
BASF has developed a Metabolomics database (MetaMap(®) Tox) containing approximately 500 data rich chemicals, agrochemicals and drugs. This metabolome-database has been built based upon 28-day studies in rats (adapted to OECD 407 guideline) with blood sampling and metabolic profiling after 7, 14 and 28 days of test substance treatment. Numerous metabolome patterns have been established for different toxicological targets (liver, kidney, thyroid, testes, blood, nervous system and endocrine system) which are specific for different toxicological modes of action. With these patterns early detection of toxicological effects and the underlying mechanism can now be obtained from routine studies. Early recognition of toxicological mode of action will help to develop new compounds with a more favourable toxicological profile and will also help to reduce the number of animal studies necessary to do so. Thus this technology contributes to animal welfare by means of reduction through refinement (2R), but also has potential as a replacement method by analyzing samples from in vitro studies. With respect to the REACH legislation for which a large number of animal studies will need to be performed, one of the most promising methods to reduce the number of animal experiments is grouping of chemicals and read-across to those which are data rich. So far mostly chemical similarity or QSAR models are driving the selection process of chemical grouping. However, "omics" technologies such as metabolomics may help to optimize the chemical grouping process by providing biologically based criteria for toxicological equivalence. "From QSAR to QBAR" (quantitative biological activity relationship).
Assuntos
Metabolômica , Relação Quantitativa Estrutura-Atividade , Toxicologia/métodos , Animais , Fígado/efeitos dos fármacos , Masculino , Modelos Teóricos , Noxas/classificação , Ratos , Glândula Tireoide/efeitos dos fármacos , Toxicologia/legislação & jurisprudênciaRESUMO
Control of mRNA stability is critical for expression of short-lived transcripts from cytokines and proto-oncogenes. Regulation involves an AU-rich element (ARE) in the 3' untranslated region (3'UTR) and cognate trans-acting factors thought to promote either degradation or stabilization of the mRNA. In this study we present a novel approach using somatic cell genetics designed to identify regulators of interleukin-3 (IL-3) mRNA turnover. Mutant cell lines were generated from diploid HT1080 cells transfected with a reporter construct containing green fluorescent protein (GFP) linked to the IL-3 3'UTR. GFP was expressed at low levels due to rapid decay of the mRNA. Following chemical mutagenesis and selection of GFP-overexpressing cells, we could isolate three mutant clones (slowA, slowB, and slowC) with a specific, trans-acting defect in IL-3 mRNA degradation, while the stability of IL-2 and tumor necrosis factor alpha reporter transcripts was not affected. Somatic cell fusion experiments revealed that the mutants are genetically recessive and form two complementation groups. Expression of the tristetraprolin gene in both groups led to reversion of the mutant phenotype, thereby linking this gene to the IL-3 mRNA degradation pathway. The genetic approach described here should allow identification of the defective functions by gene transfer and is also applicable to the study of other mRNA turnover pathways.
Assuntos
Proteínas de Ligação a DNA , Proteínas Imediatamente Precoces , Interleucina-3/genética , Proteínas/metabolismo , Estabilidade de RNA/genética , RNA Mensageiro/metabolismo , Dedos de Zinco , Animais , Genes Reporter , Teste de Complementação Genética , Humanos , Camundongos , Mutagênese , Fenótipo , Proteínas/genética , Tristetraprolina , Células Tumorais CultivadasRESUMO
AU-rich elements (ARE) present in the 3' untranslated regions of many cytokines and immediate-early genes are responsible for targeting the transcripts for rapid decay. We present evidence from cotransfection experiments in NIH 3T3 cells that two signaling pathways, one involving phosphatidylinositol 3-kinase (PI3-K), and one involving the p38 mitogen-activated protein kinase (MAPK), lead to stabilization of interleukin-3 mRNA in parallel. Stabilization mediated by either of the two pathways was antagonized by tristetraprolin (TTP), an AU-binding protein known to promote constitutive decay of ARE-containing transcripts. Remarkably, the stabilizing AU-binding protein HuR, in collaboration with p38 MAPK but not with PI3-K, could overcome the destabilizing effect of TTP. These data argue that the stabilizing kinases PI3-K and p38 MAPK do not act through direct inactivation of TTP but via activating pathway-specific stabilizing AU-binding proteins. Our data suggest an integrated model of mRNA turnover control, where stabilizing (HuR) and destabilizing (TTP) AU-binding proteins compete and where the former are under the positive control of independent phosphokinase signaling pathways.
Assuntos
Antígenos de Superfície , Proteínas de Ligação a DNA , Regulação da Expressão Gênica , Interleucina-3/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , RNA Mensageiro , Transdução de Sinais , Células 3T3 , Animais , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Proteínas Imediatamente Precoces/fisiologia , Camundongos , Proteínas de Ligação a RNA/fisiologia , Transfecção , Tristetraprolina , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Will metabolomics have a greater chance of success in toxicology and biomarker assessment than genomics and proteomics? Metabolomics has the advantage that (1) it analyses the last step in a series of changes following a toxic insult, (2) many of the metabolites have a known function and (3) changes are detectable in blood. If the analysis of a great number of individual organs can be replaced by one matrix then this will provide significant advantages (less invasive method, no need to kill animals, time course analysis possible). We have chosen to perform the analysis of blood metabolites in such a way as to minimize the risk of artifacts and to have a high number of known metabolites. We have also reduced the amount of variation in the biological system as well as during analysis. In a series of proof of concept studies it could be demonstrated that (1) the metabolome of control animals was stable of a period of nearly 1 year, with a remarkable differentiation between males and females, (2) a dose response relationship in metabolome changes was induced by phenobarbital and that (3) different modes of action could be distinguished by blood metabolome analysis. To investigate the potential of metabolomics to find biomarkers or specific patterns of change we have analyzed the blood metabolome of rats treated with HPPD inhibitors, a novel class of herbicides. The results demonstrated that a single metabolite, tyrosine, can be used as a biomarker. In addition to tyrosine we also found a specific pattern of change that involved nine metabolites. Though the extent of change was less than for tyrosine the consistent change of these metabolites is diagnostic for this (toxicological) mode of action.
Assuntos
Biomarcadores/sangue , Redes e Vias Metabólicas/efeitos dos fármacos , Biologia de Sistemas , Toxicologia/métodos , 4-Hidroxifenilpiruvato Dioxigenase/antagonistas & inibidores , 4-Hidroxifenilpiruvato Dioxigenase/metabolismo , Antagonistas de Androgênios/toxicidade , Animais , Antitireóideos/toxicidade , Cromatografia Líquida , Relação Dose-Resposta a Droga , Indução Enzimática , Inibidores Enzimáticos/toxicidade , Feminino , Flutamida/toxicidade , Cromatografia Gasosa-Espectrometria de Massas , Herbicidas/toxicidade , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Fenobarbital/toxicidade , Propiltiouracila/toxicidade , Ratos , Ratos Wistar , Reprodutibilidade dos Testes , Fatores Sexuais , Espectrometria de Massas em Tandem , Fatores de Tempo , Toxicologia/normas , Tirosina/sangueRESUMO
The MetaMap(®)-Tox database contains plasma-metabolome and toxicity data of rats obtained from oral administration of 550 reference compounds following a standardized adapted OECD 407 protocol. Here, metabolic profiles for aniline (A), chloroform (CL), ethylbenzene (EB), 2-methoxyethanol (ME), N,N-dimethylformamide (DMF) and tetrahydrofurane (THF), dosed inhalatively for six hours/day, five days a week for 4 weeks were compared to oral dosing performed daily for 4 weeks. To investigate if the oral and inhalative metabolome would be comparable statistical analyses were performed. Best correlations for metabolome changes via both routes of exposure were observed for toxicants that induced profound metabolome changes. e.g. CL and ME. Liver and testes were correctly identified as target organs. In contrast, route of exposure dependent differences in metabolic profiles were noted for low profile strength e.g. female rats dosed inhalatively with A or THF. Taken together, the current investigations demonstrate that plasma metabolome changes are generally comparable for systemic effects after oral and inhalation exposure. Differences may result from kinetics and first pass effects. For compounds inducing only weak changes, the differences between both routes of exposure are visible in the metabolome.
Assuntos
Compostos de Anilina/toxicidade , Derivados de Benzeno/toxicidade , Clorofórmio/toxicidade , Dimetilformamida/toxicidade , Etilenoglicóis/toxicidade , Furanos/toxicidade , Metaboloma , Metabolômica , Testes de Toxicidade , Administração por Inalação , Administração Oral , Compostos de Anilina/administração & dosagem , Compostos de Anilina/farmacocinética , Animais , Derivados de Benzeno/administração & dosagem , Derivados de Benzeno/farmacocinética , Clorofórmio/administração & dosagem , Clorofórmio/farmacocinética , Bases de Dados Factuais , Dimetilformamida/administração & dosagem , Dimetilformamida/farmacocinética , Relação Dose-Resposta a Droga , Esquema de Medicação , Etilenoglicóis/administração & dosagem , Etilenoglicóis/farmacocinética , Feminino , Furanos/administração & dosagem , Furanos/farmacocinética , Exposição por Inalação , Masculino , Análise de Componente Principal , Ratos Wistar , Medição de RiscoRESUMO
Changes in behavior of rats caused by different doses of cyclazocine (0.1, 0.4, 0.75, 1.5, and 3.0 mg/kg) were detected by two different methods: the open field and the residential maze. In the residential maze the locomotion was recorded automatically, whereas in the open field the measurements were made by direct observation. In the maze low doses of cyclazocine (less than 1.5 mg/kg) caused a marked change in the time course of locomotion and local activity at the beginning of the 23-h sessions. The duration of this effect was dose-dependent, between 2 and 4 h. The highest dose (3 mg/kg) induced a strong stimulation of locomotor activity which lasted about 1 h, and stereotyped patterns, i.e., long periods of unidirectional runs through circular alleys. In the open field rearing and grooming behavior proved to be the most sensitive parameters. The frequency of both was reduced at a dose of 0.4 mg/kg. Locomotion showed the highest values at 1.5 mg/kg and decreased with the highest dose (3 mg/kg) to control levels. The study demonstrated that the principal changes induced by cyclazocine were of a qualitative nature, characterized by monotonous locomotor activity. The computerized residential maze procedure proved to be well suited to detect and quantify this behavioral change.
Assuntos
Comportamento Animal/efeitos dos fármacos , Ciclazocina/farmacologia , Animais , Humanos , Masculino , Atividade Motora/efeitos dos fármacos , Ratos , Ratos Endogâmicos , Comportamento Estereotipado/efeitos dos fármacosRESUMO
The understanding of the global environmental multiphase distribution of persistent organic pollutants (POPs) as a result of the physico-chemical properties of the respective compounds is well established. We have analysed the results of a vertical transport of POPs from upper water layers (0-200 m) to the deepwater region (> 800 m) in terms of the contamination of the biophase in both water layers. The contents of persistent organochlorine compounds like polychlorinated biphenyls (PCBs) in fish living in the upper water layers of the North Atlantic and the South Atlantic, and at the continental shelf of California (Marine Sanctuary Monterey Bay and its deep-sea Canyon) are compared to the levels in deep-sea or bottom dwelling fish within the same geographic area. The deep-sea biota show significantly higher burdens as compared to surface-living species of the same region. There are also indications for recycling processes of POPs--in this case the PCBs--in the biophase of the abyss as well. It can be concluded that the bio- and geo phase of the deep-sea may act similarly as the upper horizons of forest and grasslands on the continents as an ultimate global sink for POPs in the marine environment.
Assuntos
Peixes , Bifenilos Policlorados/análise , Poluentes Químicos da Água/análise , Animais , Oceano Atlântico , California , Ecossistema , Monitoramento Ambiental/métodosRESUMO
The understanding of the global environmental multiphase distribution of persistent organic pollutants (POPs) as a result of the physico-chemical properties of the respective compounds is well established. We have analysed the results of a vertical transport of POPs from surface water to deepwater in terms of the contamination of the biota living in the respective environmental compartments. Samples were taken from the North and the South Atlantic and from the uprising water region of the continental shelf of California (Marine Sanctuary Monterey Bay and its Canyon). The contents of persistent organochlorine pesticides (DDTs, chlordanes, toxaphenes, HCHs, and HCB) in surface-living fish are compared to those in deepwater fish of the same geographic area. The deepwater biota show significantly higher burdens as compared to surface-living species of the same region. There are also indications for recycling processes of POPs of the class of organochlorine pesticides in the biophase of the abyss as well. It can be concluded that the bio- and geophase of the deep-sea may act as an ultimate global sink for persistent semivolatile contaminants in the marine environment like the soil on the continents.
Assuntos
Peixes , Hidrocarbonetos Clorados/análise , Praguicidas/análise , Poluentes Químicos da Água/análise , Animais , Oceano Atlântico , California , DDT/análise , Ecossistema , Monitoramento AmbientalRESUMO
For identification of toxicological modes of action (MoAs) a database (MetaMap(®)Tox) was established containing plasma metabolome consisting of approximately 300 endogenous metabolites. Each five male and female Wistar rats per groups were treated with >500 reference compounds over a period of 28 days. More than 120 specific toxicity patterns of common metabolite changes associated with unique MoAs were established. To establish patterns predictive effects on the thyroid, animals have been treated with reference compounds directly acting on the thyroid hormone formation (such as methimazole, ethylenethiourea) as well as liver enzyme inducers leading to an increased excretion of thyroid hormones and therewith to a secondary response of the thyroid (such as aroclor 1254 and boscalid). Here we present the plasma metabolite changes which form the patterns for direct and indirect effects on the thyroid. It is possible to identify metabolites which are commonly regulated irrespective of an indirect or direct effect on the thyroid as well as groups of metabolites separating both MoAs. By putting the metabolite regulations in the context of affected pathways helps to identify thyroid hormone inhibiting MoAs even when the hormone levels are not consistently changed. E.g., direct thyroid hormone synthesis inhibitors affect some enzymes in the urea cycle, increase the ω-oxidation of fatty acids and decrease glutamate and oxoproline levels, whereas indirect thyroid hormone inhibiting compounds interact with the lipid mediated and liver metabolism.
Assuntos
Antitireóideos/administração & dosagem , Metabolômica/métodos , Glândula Tireoide/efeitos dos fármacos , Hormônios Tireóideos/sangue , Animais , Compostos de Bifenilo/administração & dosagem , Etilenotioureia/administração & dosagem , Feminino , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Metaboloma/efeitos dos fármacos , Metimazol/administração & dosagem , Niacinamida/administração & dosagem , Niacinamida/análogos & derivados , Ratos , Ratos Wistar , Glândula Tireoide/metabolismoRESUMO
The identification of the no observed adverse effect level (NOAEL) is the key regulatory outcome of toxicity studies. With the introduction of "omics" technologies into toxicological research, the question arises as to how sensitive these technologies are relative to classical regulatory toxicity parameters. BASF SE and metanomics developed the in vivo metabolome database MetaMap®Tox containing metabolome data for more than 500 reference compounds. For several years metabolome analysis has been routinely performed in regulatory toxicity studies (REACH mandated testing or new compound development), mostly in the context of 28 day studies in rats (OECD 407 guideline). For those chemicals for which a toxicological NOAEL level was obtained at either high or mid-dose level, we evaluated the associated metabolome to investigate the sensitivity of metabolomics versus classical toxicology with respect to the NOAEL. For the definition of a metabolomics NOAEL the ECETOC criteria (ECETOC, 2007) were used. In this context we evaluated 104 cases. Comparable sensitivity was noted in 75% of the cases, increased sensitivity of metabolomics in 8%, and decreased sensitivity in 18% of the cases. In conclusion, these data suggest that metabolomics profiling has a similar sensitivity to the classical toxicological study (e.g. OECD 407) design.
Assuntos
Agroquímicos/toxicidade , Avaliação Pré-Clínica de Medicamentos , Drogas em Investigação/efeitos adversos , Metabolômica/métodos , Modelos Biológicos , Testes de Toxicidade , Agroquímicos/análise , Agroquímicos/farmacocinética , Animais , Bases de Dados de Compostos Químicos , Avaliação Pré-Clínica de Medicamentos/normas , Drogas em Investigação/análise , Drogas em Investigação/farmacocinética , Feminino , Alemanha , Guias como Assunto , Humanos , Legislação de Medicamentos , Masculino , Nível de Efeito Adverso não Observado , Medicamentos sem Prescrição/efeitos adversos , Medicamentos sem Prescrição/análise , Medicamentos sem Prescrição/farmacocinética , Medicamentos sob Prescrição/efeitos adversos , Medicamentos sob Prescrição/análise , Medicamentos sob Prescrição/farmacocinética , Ratos , Ratos Wistar , Reprodutibilidade dos Testes , Caracteres Sexuais , Testes de Toxicidade/normas , Toxicologia/legislação & jurisprudência , Toxicologia/métodosRESUMO
While conventional parameters used to detect hepatotoxicity in drug safety assessment studies are generally informative, the need remains for parameters that can detect the potential for hepatotoxicity at lower doses and/or at earlier time points. Previous work has shown that metabolite profiling (metabonomics/metabolomics) can detect signals of potential hepatotoxicity in rats treated with doxorubicin at doses that do not elicit hepatotoxicity as monitored with conventional parameters. The current study extended this observation to the question of whether such signals could be detected in rats treated with compounds that can elicit hepatotoxicity in humans (i.e., drug-induced liver injury, DILI) but have not been reported to do so in rats. Nine compounds were selected on the basis of their known DILI potential, with six other compounds chosen as negative for DILI potential. A database of rat plasma metabolite profiles, MetaMap(®)Tox (developed by metanomics GmbH and BASF SE) was used for both metabolite profiles and mode of action (MoA) metabolite signatures for a number of known toxicities. Eight of the nine compounds with DILI potential elicited metabolite profiles that matched with MoA patterns of various rat liver toxicities, including cholestasis, oxidative stress, acetaminophen-type toxicity and peroxisome proliferation. By contrast, only one of the six non-DILI compounds showed a weak match with rat liver toxicity. These results suggest that metabolite profiling may indeed have promise to detect signals of hepatotoxicity in rats treated with compounds having DILI potential.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/sangue , Doença Hepática Induzida por Substâncias e Drogas/diagnóstico , Fígado/efeitos dos fármacos , Metabolômica/métodos , Animais , Atropina/toxicidade , Captopril/toxicidade , Relação Dose-Resposta a Droga , Feminino , Flutamida/toxicidade , Lamivudina/toxicidade , Fígado/metabolismo , Masculino , Manitol/toxicidade , Metotrexato/toxicidade , Neomicina/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Fenitoína/toxicidade , Piperazinas , Propiltiouracila/toxicidade , Ratos , Ratos Wistar , Estreptomicina/toxicidade , Triazóis/toxicidade , Ácido Valproico/toxicidade , Vancomicina/toxicidade , Zidovudina/toxicidadeRESUMO
Addressing safety concerns such as drug-induced kidney injury (DIKI) early in the drug pharmaceutical development process ensures both patient safety and efficient clinical development. We describe a unique adjunct to standard safety assessment wherein the metabolite profile of treated animals is compared with the MetaMap Tox metabolomics database in order to predict the potential for a wide variety of adverse events, including DIKI. To examine this approach, a study of five compounds (phenytoin, cyclosporin A, doxorubicin, captopril, and lisinopril) was initiated by the Technology Evaluation Consortium under the auspices of the Drug Safety Executive Council (DSEC). The metabolite profiles for rats treated with these compounds matched established reference patterns in the MetaMap Tox metabolomics database indicative of each compound's well-described clinical toxicities. For example, the DIKI associated with cyclosporine A and doxorubicin was correctly predicted by metabolite profiling, while no evidence for DIKI was found for phenytoin, consistent with its clinical picture. In some cases the clinical toxicity (hepatotoxicity), not generally seen in animal studies, was detected with MetaMap Tox. Thus metabolite profiling coupled with the MetaMap Tox metabolomics database offers a unique and powerful approach for augmenting safety assessment and avoiding clinical adverse events such as DIKI.
Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/sangue , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/diagnóstico , Nefropatias/sangue , Nefropatias/induzido quimicamente , Metaboloma , Metabolômica/métodos , Animais , Captopril/efeitos adversos , Ciclosporina/efeitos adversos , Doxorrubicina/efeitos adversos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/metabolismo , Feminino , Humanos , Nefropatias/metabolismo , Lisinopril/efeitos adversos , Masculino , Fenitoína/efeitos adversos , Ratos , Ratos WistarRESUMO
BASF has developed a rat plasma metabolomics database (MetaMap®Tox) containing the metabolome of more than 500 chemicals, agrochemicals and drugs, for which the toxicity is well known, derived from 28-day repeated dose toxicity studies in rats. The quality/reproducibility of data was assessed by comparing the metabolome of 16 reference compounds tested at least twice under identical experimental conditions at three time points (day 7, day 14 and day 28). Statistical correlation analysis showed that the repeated treatment induced very similar changes to the metabolome. For all repetitions the modes of action of the compounds were always correctly identified. Moreover, when compared against the metabolome of all compounds available in the MetaMap®Tox database, the repetitions showed in most cases the highest degree of overall similarity with the metabolome of the original study. In addition, we also evaluated the robustness of our metabolomics technique, displayed by constancy of variability in control groups over time. Based on these results, it can be concluded, that metabolomics can reproducibly be applied during toxicological in vivo testing in rats under the conditions applied here.
Assuntos
Análise Química do Sangue/métodos , Metabolômica/métodos , Testes de Toxicidade/métodos , Animais , Análise Química do Sangue/instrumentação , Feminino , Masculino , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Metaboloma , Metabolômica/normas , Ratos , Ratos Endogâmicos WF , Estatísticas não Paramétricas , Testes de Toxicidade/normasRESUMO
Combination therapies with fibrates and statins are used to treat cardiovascular diseases, because of their synergistic effect on lowering plasma lipids. However, fatal side-effects like rhabdomyolysis followed by acute renal necrosis sometimes occur. To elucidate biochemical changes resulting from the interaction of fibrates and statins, doses of 100 mg/kg fenofibrate, 50mg/kg clofibrate, 70 mg/kg atorvastatin and 200 mg/kg pravastatin as well as combinations thereof were administered to Crl:Wi(Han) rats for 4 weeks. Plasma metabolome profile was measured on study days 7, 14 and 28. Upon study termination, clinical pathology parameters were measured. In a separate experiment plasmakinetic data were measured in male rats after 1 week of drug administration in monotherapy as well as in combinations. Lowering of blood lipid levels as well as toxicological effects, like liver cell degradation (statins) and anemia (fibrates) and distinct blood metabolite level alterations were observed in monotherapy. When fibrates and statins were co-administered metabolite profile interactions were generally underadditive or at the utmost additive according to the linear mixed effect model. However, more metabolite levels were significantly altered during combination therapy. New effects on the antioxidant status and the cardiovascular system were found which may be related to a development of rhabdomyolysis. Accumulation of drugs during the combination therapy was not observed.
Assuntos
Doenças Cardiovasculares/tratamento farmacológico , Ácidos Fíbricos/toxicidade , Inibidores de Hidroximetilglutaril-CoA Redutases/toxicidade , Hipolipemiantes/toxicidade , Animais , Contagem de Células Sanguíneas , Peso Corporal/efeitos dos fármacos , Doenças Cardiovasculares/metabolismo , Quimioterapia Combinada/efeitos adversos , Ingestão de Alimentos/efeitos dos fármacos , Ácidos Fíbricos/administração & dosagem , Ácidos Fíbricos/farmacocinética , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacocinética , Hipolipemiantes/administração & dosagem , Hipolipemiantes/farmacocinética , Masculino , Metabolômica/métodos , Ratos , Espectrometria de Massas em TandemRESUMO
BASF and Metanomics have built-up the database MetaMap(®)-Tox containing rat plasma metabolome data for more than 500 reference compounds. Phenytoin was administered to five Wistar rats of both sexes at dietary dose levels of 600 and 2400 ppm over 28 days and metabolome analysis was performed on days 7, 14 and 28. Clinical pathology did not indicate clear evidence for liver toxicity, whereas liver histopathology revealed slight centrilobular hepatocellular hypertrophy. The metabolome analysis of phenytoin shows metabolome changes at both dose levels and the comparison with MetaMap-Tox indicated strong evidence for liver enzyme induction, as well as liver toxicity. Moreover, evidence for kidney and indirect thyroid effects were observed. This assessment was based on the metabolite changes induced, similarities to specific toxicity patterns and the whole metabolome correlation within MetaMap-Tox. As compared with the classical read-out, a more comprehensive picture of phenytoin's effects is obtained from the metabolome analysis, demonstrating the added value of metabolome data in preclinical/ toxicological studies.
Assuntos
Biomarcadores Farmacológicos/sangue , Metaboloma/efeitos dos fármacos , Metabolômica/métodos , Fenitoína/toxicidade , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Cromatografia Gasosa-Espectrometria de Massas/métodos , Fígado/patologia , Masculino , Fenitoína/administração & dosagem , Ratos , Ratos Wistar , Espectrometria de Massas em Tandem/métodosRESUMO
The study presented here describes the application of metabolite profiling of highly polar, intracellular metabolites after incubation of a mammalian fibroblast cell line with inhibitors of mitochondrial function. A metabolomics approach was used to assess the complex response of the cellular energy metabolism. Metabolic profiles of phosphorylated and carboxylated intracellular metabolites were assessed by UPLC-MS/MS and used to predict the mode of mitochondrial toxicity. Based on distinct metabolic patterns, multivariate data analysis allowed for the discrimination of two groups of toxins: inhibitors of the electron transport in mitochondrial membranes (complex IV inhibitors) and uncouplers of oxidative phosphorylation. Beyond these known interferences, metabolic profiling was able to reveal additional inhibitory effects on the cellular energy metabolism. Most prominently, for three of the toxins, metabolic patterns also disclosed an enhanced activity of the glycerol phosphate shuttle inferring the inhibition of NADH dehydrogenase at complex I. Secondly, inhibition of the electron transport was accompanied by a limiting availability of citric acid cycle intermediates and aspartate. Concomitantly, specific perturbations of the purine nucleotide cycle were observed. We have shown here that metabolomic approaches may assist to predict complex modes of action of toxic compounds on cellular level as well as to unravel specific dysfunctions in the energy metabolism.
Assuntos
Metabolismo Energético , Metabolômica , Mitocôndrias/metabolismo , 2,4-Dinitrofenol/farmacologia , Animais , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Células Cultivadas , Cricetinae , Cricetulus , Transporte de Elétrons/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Cianeto de Potássio/farmacologia , Azida Sódica/farmacologiaRESUMO
Metabolite profiling (metabolomics) elucidates changes in biochemical pathways under various conditions, e.g., different nutrition scenarios or compound administration. BASF and metanomics have obtained plasma metabolic profiles of approximately 500 compounds (agrochemicals, chemicals and pharmaceuticals) from 28-day rat studies. With these profiles the establishment of a database (MetaMap(®)Tox) containing specific metabolic patterns associated with many toxicological modes of action was achieved. To evaluate confounding factors influencing metabolome patterns, the effect of fasting vs. non-fasting prior to blood sampling, the influence of high caloric diet and caloric restriction as well as the administration of corn oil and olive oil was studied for its influence on the metabolome. All mentioned treatments had distinct effects: triacylglycerol, phospholipids and their degradation product levels (fatty acids, glycerol, lysophosphatidylcholine) were often altered depending on the nutritional status. Also some amino acid and related compounds were changed. Some metabolites derived from food (e.g. alpha-tocopherol, ascorbic acid, beta-sitosterol, campesterol) were biomarkers related to food consumption, whereas others indicated a changed energy metabolism (e.g. hydroxybutyrate, pyruvate). Strikingly, there was a profound difference in the metabolite responses to diet restriction in male and female rats. Consequently, when evaluating the metabolic profile of a compound, the effect of nutritional status should be taken into account.
Assuntos
Fenômenos Fisiológicos da Nutrição Animal/fisiologia , Análise Química do Sangue , Metaboloma/fisiologia , Fenômenos Fisiológicos da Nutrição Animal/efeitos dos fármacos , Animais , Restrição Calórica , Óleo de Milho/farmacologia , Dieta , Jejum/sangue , Jejum/fisiologia , Feminino , Masculino , Metaboloma/efeitos dos fármacos , Estado Nutricional , Azeite de Oliva , Óleos de Plantas/farmacologia , Ratos , Ratos WistarRESUMO
Metabolite profiles (metabolomics) of plasma samples of Wistar rats dosed with di(2-ethylhexyl)phthalate (DEHP - 3000ppm) and dibutylphthalate (DBP - 150, 1000 and 7000ppm) were individually determined in 28 days dietary studies. In addition, profiles of combined exposure to 3000ppm DEHP and either 150, 1000 or 7000ppm DBP were determined. High dose levels induced more profound metabolite changes in males than in females for both compounds. At 150ppm DBP (NOEL for toxicity) there were very few (Assuntos
Dibutilftalato/toxicidade
, Dietilexilftalato/toxicidade
, Poluentes Ambientais/toxicidade
, Metaboloma/efeitos dos fármacos
, Administração Oral
, Animais
, Biomarcadores/sangue
, Cromatografia Líquida de Alta Pressão
, Relação Dose-Resposta a Droga
, Sinergismo Farmacológico
, Feminino
, Cromatografia Gasosa-Espectrometria de Massas
, Fígado/efeitos dos fármacos
, Fígado/metabolismo
, Fígado/patologia
, Masculino
, Metabolômica/métodos
, Tamanho do Órgão/efeitos dos fármacos
, Ratos
, Ratos Wistar
, Fatores Sexuais
, Espectrometria de Massas em Tandem
, Testes de Toxicidade Crônica
Assuntos
Chaperonina 60/isolamento & purificação , Mitocôndrias/metabolismo , Pichia/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia por Troca Iônica/métodos , Citosol/metabolismo , Proteínas Fúngicas/isolamento & purificação , Indicadores e Reagentes , Pichia/crescimento & desenvolvimentoRESUMO
The impact of the strain on the metabolite profile of plasma samples in rats dosed with 2500 ppm 2-methyl-4-chlorophenoxyacetic acid (MCPA acid) or 45 mg/kg bw/day 4-chloro-3-nitroaniline (4C3N) for 4 weeks was evaluated. Four different strains were used: two Wistar strains (Crl:WI(Han), Han:RCC:WIST(SPF)), one Sprague-Dawley (Crl:CD) and one Fisher strain (F-344/Crl). The metabolite profiles in the plasma were measured by LC-MS and GC-MS. The profound changes of the metabolite values induced by the MCPA acid treatment outweighed slight deviations caused by physiological variations between the different rat strains. The metabolome changes of the MCPA acid in all strains could be related to toxicological "mode of action" patterns (peroxisome proliferator, renal organic anionic transporter inhibition) with Crl:WI(Han) rats as reference strain. 4C3N administration led to extravascular hemolytic anemia with a small number of metabolome changes, which were strain dependent. The metabolome pattern associated with "hemolytic anemia" established with the reference strain (Crl:Wi(Han)) was not sufficiently similar in other strains. Thus, comparable metabolome profiles were obtained in different rat strains for a compound inducing profound metabolite changes. For a compound with a weak profile the results were more variable and appeared to be strain dependent.