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1.
Mol Cell Neurosci ; 71: 92-101, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26711807

RESUMO

Parkinson's disease is characterized by degeneration of dopaminergic neurons in the substantia nigra pars compacta along with the formation of intracellular fibrillar inclusions (Lewy bodies and Lewy neuritis), which are mainly composed of aggregated α-synuclein (ASYN). This latter is a 14 kDa protein that localizes to synaptic vesicles in nerve terminals and promotes soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex assembly. We explored the monomeric and oligomeric state of ASYN in vitro in HEK293s and SH-SY5Y cell lines. In addition rats were injected in the substantia nigra with an Adeno associated virus carrying the human A53T mutation of ASYN (in vivo experiments). We show that human wild type ASYN as well as PD-linked mutations (A30P, E46K and A53T) in overexpressing conditions mostly exists in a monomeric state in equilibrium with dimeric forms. The monomer/dimer ratio is unaffected by PD-linked mutation. Furthermore, the A30P, E46K and A53T mutations overexpression strongly increased cell death compared to wild type ASYN. Taken together, our data suggest that ASYN dimers amount do not directly correlate to reduced cellular viability, suggesting a different role in protein function and induced pathology. Our data suggest that early ASYN neuro-pathogenic effects are probably mediated by other molecular processes than increased oligomerization alone.


Assuntos
Doença de Parkinson/metabolismo , Multimerização Proteica , alfa-Sinucleína/metabolismo , Animais , Morte Celular , Linhagem Celular Tumoral , Neurônios Dopaminérgicos/metabolismo , Células HEK293 , Humanos , Doença de Parkinson/genética , Mutação Puntual , Ratos , Ratos Sprague-Dawley , Substância Negra/metabolismo , Substância Negra/patologia , alfa-Sinucleína/química , alfa-Sinucleína/genética
2.
Mutat Res ; 751(1): 1-11, 2013 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-23159395

RESUMO

Recent publications on the automated in vitro micronucleus assay show predictive values higher than 85% for the classification of in vitro aneugens, clastogens and non-genotoxic compounds. In the present work, the CHO-k1 micronucleus assay in combination with cellular imaging was further evaluated. Firstly, the effect of a range of S9 concentrations on micronucleus formation and cytotoxicity was investigated. Subsequently, the reproducibility and predictivity of the micronucleus assay on CHO-k1 cells was investigated with a set of four compounds. Then, a larger set of compounds (n=44) was tested on CHO-k1 cells and inter-laboratory correlation was calculated. Finally, cellular imaging was compared with flow cytometry for in vivo assessment of micronucleus formation. The concentration of S9 had a significant impact on micronucleus formation and cytotoxicity. In addition, calculations of relative cell count (RCC) and cytokinesis-block proliferation index (CBPI) showed to be complementary to cytotoxicity assessment. The CHO-k1 micronucleus assay correctly classified the four reference compounds, with a dose-response relationship and low variability. Based on a larger set of compounds, the assay proved to be reliable with a sensitivity of 94% (n=31) and a specificity of 85% (n=13). A correlation coefficient of 97% was obtained when the lowest observable adverse effect levels (LOAELs) from our study were compared with those published by Diaz et al. (2007) [10]. In conclusion, the in vitro CHO-k1 micronucleus assay combined with cellular imaging is a predictive assay appropriate for genotoxicity screening at early stages of drug development. In addition, for in vivo assessment of micronucleus formation, we preferred to use flow cytometry rather than cell imaging.


Assuntos
Aneugênicos/toxicidade , Processamento de Imagem Assistida por Computador , Testes para Micronúcleos/métodos , Animais , Biotransformação , Células CHO , Contagem de Células , Cricetinae , Cricetulus , Citocinese , Relação Dose-Resposta a Droga , Descoberta de Drogas , Citometria de Fluxo , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
3.
Cell Biol Toxicol ; 28(2): 69-87, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22258563

RESUMO

In the pharmaceutical industry, improving the early detection of drug-induced hepatotoxicity is essential as it is one of the most important reasons for attrition of candidate drugs during the later stages of drug development. The first objective of this study was to better characterize different cellular models (i.e., HepG2, HepaRG cells, and fresh primary human hepatocytes) at the gene expression level and analyze their metabolic cytochrome P450 capabilities. The cellular models were exposed to three different CYP450 inducers; beta-naphthoflavone (BNF), phenobarbital (PB), and rifampicin (RIF). HepG2 cells responded very weakly to the different inducers at the gene expression level, and this translated generally into low CYP450 activities in the induced cells compared with the control cells. On the contrary, HepaRG cells and the three human donors were inducible after exposure to BNF, PB, and RIF according to gene expression responses and CYP450 activities. Consequently, HepaRG cells could be used in screening as a substitute and/or in complement to primary hepatocytes for CYP induction studies. The second objective was to investigate the predictivity of the different cellular models to detect hepatotoxins (16 hepatotoxic and 5 nonhepatotoxic compounds). Specificity was 100% with the different cellular models tested. Cryopreserved human hepatocytes gave the highest sensitivity, ranging from 31% to 44% (depending on the donor), followed by lower sensitivity (13%) for HepaRG and HepG2 cells (6.3%). Overall, none of the models under study gave desirable sensitivities (80-100%). Consequently, a high metabolic capacity and CYP inducibility in cell lines does not necessarily correlate with a high sensitivity for the detection of hepatotoxic drugs. Further investigations are necessary to compare different cellular models and determine those that are best suited for the detection of hepatotoxic compounds.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/etiologia , Sistema Enzimático do Citocromo P-450/metabolismo , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Hepatócitos/efeitos dos fármacos , RNA Mensageiro/genética , Testes de Toxicidade/métodos , Adulto , Idoso , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Doença Hepática Induzida por Substâncias e Drogas/genética , Criança , Sistema Enzimático do Citocromo P-450/biossíntese , Indução Enzimática , Feminino , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Células Hep G2 , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Humanos , Concentração Inibidora 50 , Dose Letal Mediana , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Valor Preditivo dos Testes , Análise de Componente Principal , Toxicogenética
4.
Toxicol In Vitro ; 23(2): 319-32, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19110050

RESUMO

The present study was undertaken to validate a battery of cytotoxicity assays performed in a multiplex format to screen pharmaceutical compounds at an early stage of drug development. Two experiments were performed on HepG2 cells and the parameters were measured in 96-well plates. Biological and technical triplicates were performed to evaluate the reproducibility of the assay. In the first experiment, HepG2 cells were exposed to tamoxifen, staurosporine, phenobarbital and triton X-100 for 2 and 24h. The following nine cytotoxicity parameters were analyzed, cell viability, lactate dehydrogenase (LDH), adenosine triphosphate (ATP), caspase-3/7, aspartate aminotransferase (AST), glutamate dehydrogenase (GLDH), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and alpha-glutathione-S-transferase (alpha-GST). In the second experiment, HepG2 cells were exposed to doxorubicin, t-butyl hydroperoxide, ferrous sulfate and sulfamoxole for 2 and 24h. Based on the results of the first experiment, six cytotoxicity parameters were selected for further evaluation (cell viability, ATP, LDH, caspase, AST and GLDH). ALT (activity always below detection limit), ALP (no response to drug treatment) and alpha-GST (too labor intensive and not possible to multiplex) were eliminated. The analysis of the data revealed that the reproducibility of the assays was accurate according to principal component analysis. Our data also clearly indicated that the potential of this battery of selected assays measured in a multiplex format not only made it possible to rank and select the most promising drug candidates based on their cytotoxic potential, but also to gather information that may help to understand some of the toxic events occurring in the cells.


Assuntos
Biomarcadores/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Hepatócitos/efeitos dos fármacos , Xenobióticos/toxicidade , Trifosfato de Adenosina/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Enzimas/metabolismo , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Programas de Rastreamento/métodos , Preparações Farmacêuticas/classificação , Reprodutibilidade dos Testes , Xenobióticos/classificação
5.
Toxicol In Vitro ; 52: 374-383, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30030051

RESUMO

Drug-induced mitochondrial toxicity is considered as a common cellular mechanism that can induce a variety of organ toxicities. In the present manuscript, 17 in vitro mitochondrial toxic drugs, reported to induce Drug-Induced Liver Injury (DILI) and 6 non-mitochondrial toxic drugs (3 with DILI and 3 without DILI concern), were tested in HepG2 cells using a bioenergetics system. The 17 mitochondrial toxic drugs represent a wide variety of mitochondrial dysfunctions as well as DILI and include 4 pairs of drugs which are structurally related but associated with different DILI concerns in human. Cell bioenergetics were measured using the XF96e analyzer which simultaneous monitor oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), indirect measurements of oxidative phosphorylation and glycolysis, respectively. OCR associated with ATP production, maximal respiration, proton leak and spare respiratory capacity, were also assessed. Duplicate experiments resulted in a sensitivity of 82% (14/17) and specificity of 83% (5/6). The addition of stressors improved specificity considerably. Cut-offs, statistics and rules are clearly discussed to facilitate the use of this assay for screening purposes. Overall, the authors consider that this assay should be part of the battery of safety screening assays at early stages of drug development.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Metabolismo Energético/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Células Hep G2 , Humanos , Mitocôndrias/metabolismo
6.
Toxicol Sci ; 59(2): 241-50, 2001 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11158717

RESUMO

This study compared the effects occurring at molecular and population levels in Daphnia magna exposed to copper concentrations in the range of 15-120 microg/l. The qualitative and quantitative modifications arising in random amplified polymorphic DNA (RAPD) profiles as a measure of DNA effects were compared with a number of key ecological fitness parameters, namely, the age-specific survival, age-specific fecundity, net reproductive rate, and intrinsic rate of population increase. Results suggested that growth, reproduction, and most of the fitness parameters as well as genomic template stability (a qualitative measure reflecting changes in RAPD profiles) were significantly affected at copper concentrations of 90 and 120 microg/l. Among the fitness parameters, the age-specific fecundity and net reproductive rate were the most sensitive parameters of toxicity. Changes in RAPD patterns generally occurred at copper concentrations of 90 and 120 microg/l, but with one primer, changes significantly arose at all copper concentrations. Overall, molecular and population parameters compared well and represented a sensitive means to measure toxicity induced by copper in Daphnia magna. In conclusion, the measurement of parameters at both molecular and population levels is valuable for investigating the specific effects of agents interacting with DNA. Ultimately, this methodology may allow the ecotoxicological examination of the link between molecular alterations and measurable adverse effects at higher levels of biological organization.


Assuntos
Cobre/toxicidade , DNA/efeitos dos fármacos , Daphnia/efeitos dos fármacos , Dinâmica Populacional , Animais , DNA/análise , Daphnia/genética , Daphnia/crescimento & desenvolvimento , Relação Dose-Resposta a Droga , Genômica , Dose Letal Mediana , Técnica de Amplificação ao Acaso de DNA Polimórfico , Reprodução/efeitos dos fármacos
7.
Toxicol In Vitro ; 25(7): 1414-24, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21515356

RESUMO

Phospholipidosis (PLD) is a topic of concern in drug development because it may be associated with toxicological consequences. The aim of the study was to determine the best method to screen proprietary compounds with regard to their potential to induce PLD. Two in vitro approaches, a genomic method previously evaluated in our laboratory and a fluorescent cell based approach to detect PLD were compared using HepG2 cells. The same set of reference compounds (15 PLD inducing, 7 non-PLD inducing and 4 additional compounds) were used to compare both approaches. The same sensitivity (15/15) and similar specificity (7/7 versus, 6/7 for the genomic approach) were obtained. In addition, 11 proprietary compounds were tested in 4-day exploratory rat toxicity studies as well as in both in vitro approaches. Two of the 11 compounds induced alveolar foamy macrophages and lung vacuolization in vivo and were considered as PLD inducers. Sensitivity (2/2) and specificity (7/9) were better with the fluorescent method than with the genomic approach (1/2 and 3/9, respectively). In conclusion, compared to the genomic approach, the fluorescent method is the test of choice for screening compounds at an early stage of drug development. Indeed, the fluorescent method is more adapted to medium throughput, detects the positive reference compounds at lower (8/15) or equal (7/15) concentrations, allows multiplexing and is associated with higher sensitivity and specificity to predict lung foamy macrophages and vacuolization in vivo. Nevertheless, to confirm our conclusion, it would be necessary to compare the predictivity of both in vitro approaches by using a wide range of reference and proprietary compounds, with information on their potential to induce PLD under in vivo conditions.


Assuntos
Lipidoses/induzido quimicamente , Lipidoses/diagnóstico , Fosfolipídeos/metabolismo , Testes de Toxicidade/métodos , Amiodarona/toxicidade , Alternativas aos Testes com Animais/métodos , Animais , Antiarrítmicos/toxicidade , Corantes Fluorescentes/metabolismo , Células Espumosas/efeitos dos fármacos , Regulação da Expressão Gênica , Genômica , Células Hep G2 , Humanos , Pneumopatias/induzido quimicamente , Macrófagos Alveolares/efeitos dos fármacos , Fosfolipídeos/análise , Ratos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
8.
Appl Environ Microbiol ; 66(1): 435-8, 2000 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-10618262

RESUMO

The molecular diversity among 60 isolates of Renibacterium salmoninarum which differ in place and date of isolation was investigated by using randomly amplified polymorphic DNA (RAPD) analysis. Isolates were grouped into 21 banding patterns which did not reflect the biological source. Four 16S-23S rRNA intergenic spacer (ITS1) sequence variations and two alleles of an exact tandem repeat locus, ETR-A, were the bases for formation of distinct groups within the RAPD clusters. This study provides evidence that the most common ITS1 sequence variant, SV1, possesses two copies of a 51-bp repeat unit at ETR-A and has been widely dispersed among countries which are associated with mainstream intensive salmonid culture.


Assuntos
Variação Genética , Micrococcaceae/genética , Técnica de Amplificação ao Acaso de DNA Polimórfico , Salmonidae/microbiologia , Animais , Doenças dos Peixes/microbiologia , Infecções por Bactérias Gram-Positivas/microbiologia , Infecções por Bactérias Gram-Positivas/veterinária , Micrococcaceae/crescimento & desenvolvimento , Micrococcaceae/isolamento & purificação , Dados de Sequência Molecular , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , Sequências de Repetição em Tandem/genética
9.
Appl Environ Microbiol ; 65(3): 961-8, 1999 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10049848

RESUMO

Renibacterium salmoninarum is a genospecies that is an obligate pathogen of salmonid fish and is capable of intracellular survival. Conventional typing systems have failed to differentiate isolates of R. salmoninarum. We used two methods to assess the extent of molecular variation which was present in isolates from different geographic locations. In one analysis we investigated possible polymorphisms in a specific region of the genome, the intergenic spacer (ITS) region between the 16S and 23S rRNA genes. In the other analysis we analyzed differences throughout the genome by using randomly amplified polymorphic DNA (RAPD). We amplified the spacer region of 74 isolates by using PCR and performed a DNA sequence analysis with 14 geographically distinct samples. The results showed that the 16S-23S ribosomal DNA spacer region of R. salmoninarum is highly conserved and suggested that only a single copy of the rRNA operon is present in this slowly growing pathogen. DNA sequencing of the spacer region showed that it was the same length in all 14 isolates examined, and the same nucleotide sequence, sequevar 1, was obtained for 11 of these isolates. Two other sequevars were found. No tRNA genes were found. We found that RAPD analysis allows reproducible differentiation between isolates of R. salmoninarum obtained from different hosts and different geographic regions. By using RAPD analysis it was possible to differentiate between isolates with identical ITS sequences.


Assuntos
Doenças dos Peixes/microbiologia , Infecções por Bactérias Gram-Positivas/veterinária , Micrococcaceae/classificação , Micrococcaceae/genética , Animais , Técnicas de Tipagem Bacteriana , Sequência de Bases , DNA Bacteriano/análise , DNA Ribossômico/análise , Doenças dos Peixes/epidemiologia , Genes Bacterianos , Genes de RNAr , Infecções por Bactérias Gram-Positivas/microbiologia , Micrococcaceae/isolamento & purificação , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , Técnica de Amplificação ao Acaso de DNA Polimórfico , Análise de Sequência de DNA
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