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1.
Environ Toxicol ; 32(4): 1375-1389, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27501252

RESUMO

Pharmaceuticals are found in the environment but the impact of this contamination on human and animal health is poorly known. The liver could be particularly targeted since a significant number of these drugs are hepatotoxic, in particular via oxidative stress and mitochondrial dysfunction. Notably, the latter events can also be observed in liver diseases linked to obesity, so that the obese liver might be more sensitive to drug toxicity. In this study, we determined the effects of a chronic exposure to low doses of pharmaceuticals in wild-type and obese mice, with a particular focus on mitochondrial function. To this end, wild-type and ob/ob mice were exposed for 4 months to a cocktail of 11 pharmaceuticals provided in drinking water containing 0.01, 0.1, or 1 mg/L of each drug. At the end of the treatment, liver mitochondria were isolated and different parameters were measured. Chronic exposure to the pharmaceuticals reduced mitochondrial respiration driven by succinate and palmitoyl-l-carnitine in wild-type mice and increased antimycin-induced ROS production in ob/ob mice. Hyperglycemia and hepatic histological abnormalities were also observed in treated ob/ob mice. Investigations were also carried out in isolated liver mitochondria incubated with the mixture, or with each individual drug. The mitochondrial effects of the mixture were different from those observed in treated mice and could not be predicted from the results obtained with each drug. Because some of the 11 drugs included in our cocktail can be found in water at relatively high concentrations, our data could be relevant in environmental toxicology. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1375-1389, 2017.


Assuntos
Poluentes Ambientais/toxicidade , Hiperglicemia/induzido quimicamente , Fígado/efeitos dos fármacos , Obesidade/sangue , Animais , Glicemia , Relação Dose-Resposta a Droga , Feminino , Hiperglicemia/sangue , Fígado/metabolismo , Fígado/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Mitocôndrias Hepáticas/metabolismo , Dilatação Mitocondrial
2.
Toxicol Appl Pharmacol ; 276(1): 63-72, 2014 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-24525044

RESUMO

Drinking water can be contaminated with pharmaceuticals. However, it is uncertain whether this contamination can be harmful for the liver, especially during obesity. Hence, the goal of our study was to determine whether chronic exposure to low doses of pharmaceuticals could have deleterious effects on livers of lean and obese mice. To this end, lean and ob/ob male mice were treated for 4 months with a mixture of 11 drugs provided in drinking water at concentrations ranging from 10 to 106 ng/l. At the end of the treatment, some liver and plasma abnormalities were observed in ob/ob mice treated with the cocktail containing 106 ng/l of each drug. For this dosage, a gene expression analysis by microarray showed altered expression of circadian genes (e.g. Bmal1, Dbp, Cry1) in lean and obese mice. RT-qPCR analyses carried out in all groups of animals confirmed that expression of 8 different circadian genes was modified in a dose-dependent manner. For some genes, a significant modification was observed for dosages as low as 10²-10³ ng/l. Drug mixture and obesity presented an additive effect on circadian gene expression. These data were validated in an independent study performed in female mice. Thus, our study showed that chronic exposure to trace pharmaceuticals disturbed hepatic expression of circadian genes, particularly in obese mice. Because some of the 11 drugs can be found in drinking water at such concentrations (e.g. acetaminophen, carbamazepine, ibuprofen) our data could be relevant in environmental toxicology, especially for obese individuals exposed to these contaminants.


Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Obesidade/metabolismo , Proteínas Circadianas Period/metabolismo , Preparações Farmacêuticas/administração & dosagem , Poluentes Químicos da Água/administração & dosagem , Fatores de Transcrição ARNTL/agonistas , Fatores de Transcrição ARNTL/antagonistas & inibidores , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Animais , Criptocromos/agonistas , Criptocromos/antagonistas & inibidores , Criptocromos/genética , Criptocromos/metabolismo , Proteínas de Ligação a DNA/agonistas , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Feminino , Perfilação da Expressão Gênica , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/induzido quimicamente , Obesidade/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Circadianas Period/agonistas , Proteínas Circadianas Period/antagonistas & inibidores , Proteínas Circadianas Period/genética , Testes de Toxicidade Crônica , Fatores de Transcrição/agonistas , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Poluentes Químicos da Água/toxicidade
3.
Toxicol Sci ; 200(2): 369-381, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-38676573

RESUMO

Drug-induced liver injury (DILI) represents a major issue for pharmaceutical companies, being a potential cause of black-box warnings on marketed pharmaceuticals, or drug withdrawal from the market. Lipid accumulation in the liver also referred to as steatosis, may be secondary to impaired mitochondrial fatty acid oxidation (mtFAO). However, an overall causal relationship between drug-induced mtFAO inhibition and the occurrence of steatosis in patients has not yet been established with a high number of pharmaceuticals. Hence, 32 steatogenic and 13 nonsteatogenic drugs were tested for their ability to inhibit mtFAO in isolated mouse liver mitochondria. To this end, mitochondrial respiration was measured with palmitoyl-l-carnitine, palmitoyl-CoA + l-carnitine, or octanoyl- l-carnitine. This mtFAO tri-parametric assay was able to predict the occurrence of steatosis in patients with a sensitivity and positive predictive value above 88%. To get further information regarding the mechanism of drug-induced mtFAO impairment, mitochondrial respiration was also measured with malate/glutamate or succinate. Drugs such as diclofenac, methotrexate, and troglitazone could inhibit mtFAO secondary to an impairment of the mitochondrial respiratory chain, whereas dexamethasone, olanzapine, and zidovudine appeared to impair mtFAO directly. Mitochondrial swelling, transmembrane potential, and production of reactive oxygen species were also assessed for all compounds. Only the steatogenic drugs amiodarone, ketoconazole, lovastatin, and toremifene altered all these 3 mitochondrial parameters. In conclusion, our tri-parametric mtFAO assay could be useful in predicting the occurrence of steatosis in patients. The combination of this assay with other mitochondrial parameters could also help to better understand the mechanism of drug-induced mtFAO inhibition.


Assuntos
Ácidos Graxos , Fígado Gorduroso , Mitocôndrias Hepáticas , Oxirredução , Animais , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Ácidos Graxos/metabolismo , Fígado Gorduroso/induzido quimicamente , Fígado Gorduroso/metabolismo , Camundongos , Masculino , Camundongos Endogâmicos C57BL , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Preparações Farmacêuticas/metabolismo
4.
Zebrafish ; 17(4): 268-270, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32364833

RESUMO

In all animal species, oxygen consumption is a key process that is partially impaired in a large number of pathological situations and thus provides informative details on the physiopathology of the disease. In this study, we describe a simple and affordable method to precisely measure oxygen consumption in living zebrafish larvae using a spectrofluorometer and the MitoXpress Xtra Oxygen Consumption Assay. In addition, we used zebrafish larvae treated with mitochondrial respiratory chain inhibitors, antimycin A or rotenone, to verify that our method enables precise and reliable measurements of oxygen consumption.


Assuntos
Antimicina A/farmacologia , Embrião não Mamífero/metabolismo , Consumo de Oxigênio , Rotenona/farmacologia , Peixe-Zebra/metabolismo , Animais , Larva/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/crescimento & desenvolvimento
5.
Apoptosis ; 14(10): 1190-203, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19693674

RESUMO

Dengue viruses belong to the Flavivirus family and are responsible for hemorrhagic fever in Human. Dengue virus infection triggers apoptosis especially through the expression of the small membrane (M) protein. Using isolated mitochondria, we found that synthetic peptides containing the C-terminus part of the M ectodomain caused apoptosis-related mitochondrial membrane permeabilization (MMP) events. These events include matrix swelling and the dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)). Protein M Flavivirus sequence alignments and helical wheel projections reveal a conserved distribution of charged residues. Moreover, when combined to the cell penetrating HIV-1 Tat peptide transduction domain (Tat-PTD), this sequence triggers a caspase-dependent cell death associated with DeltaPsi(m) loss and cytochrome c release. Mutational approaches coupled to functional screening on isolated mitochondria resulted in the selection of a protein M derived sequence containing nine residues with potent MMP-inducing properties on isolated mitochondria. A chimeric peptide composed of a Tat-PTD linked to the 9-mer entity triggers MMP and cell death. Finally, local administration of this chimeric peptide induces growth inhibition of xenograft prostate PC3 tumors in immuno-compromised mice, and significantly enhances animal survival. Together, these findings support the notion of using viral genomes as valuable sources to discover mitochondria-targeted sequences that may lead to the development of new anticancer compounds.


Assuntos
Flavivirus/química , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Peptídeos/farmacologia , Proteínas Virais/química , Ensaios Antitumorais Modelo de Xenoenxerto , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Dilatação Mitocondrial/efeitos dos fármacos , Dados de Sequência Molecular , Peptídeos/química , Permeabilidade/efeitos dos fármacos , Estrutura Terciária de Proteína , Análise de Sobrevida , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacologia
6.
J Clin Med ; 6(6)2017 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-28594349

RESUMO

In the last ten years, the use of fluorescent probes developed to measure oxygen has resulted in several marketed devices, some unreasonably expensive and with little flexibility. We have explored the use of the effective, versatile, and inexpensive Redflash technology to determine oxygen uptake by a number of different biological samples using various layouts. This technology relies on the use of an optic fiber equipped at its tip with a membrane coated with a fluorescent dye (www.pyro-science.com). This oxygen-sensitive dye uses red light excitation and lifetime detection in the near infrared. So far, the use of this technology has mostly been used to determine oxygen concentration in open spaces for environmental studies, especially in aquatic media. The oxygen uptake determined by the device can be easily assessed in small volumes of respiration medium and combined with the measurement of additional parameters, such as lactate excretion by intact cells or the membrane potential of purified mitochondria. We conclude that the performance of by this technology should make it a first choice in the context of both fundamental studies and investigations for respiratory chain deficiencies in human samples.

7.
J Alzheimers Dis ; 35(1): 107-20, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23364140

RESUMO

Increased production of amyloid-ß (Aß)42 peptide, derived from the amyloid-ß protein precursor, and its subsequent aggregation into oligomers and plaques constitutes a hallmark of Alzheimer's disease (AD). We here report on a family of low molecular weight molecules, the Aftins (Amyloid-ß Forty-Two Inducers), which, in cultured cells, dramatically affect the production of extracellular/secreted amyloid peptides. Aftins trigger ß-secretase inhibitor and γ-secretase inhibitors (GSIs) sensitive, robust upregulation of Aß42, and parallel down-regulation of Aß38, while Aß40 levels remain stable. In contrast, intracellular levels of these amyloids appear to remain stable. In terms of their effects on Aß38/Aß40/Aß42 relative abundance, Aftins act opposite to γ-secretase modulators (GSMs). Aß42 upregulation induced by Aftin-5 is unlikely to originate from reduced proteolytic degradation or diminished autophagy. Aftin-5 has little effects on mitochondrial functional parameters (swelling, transmembrane potential loss, cytochrome c release, oxygen consumption) but reversibly alters the ultrastructure of mitochondria. Aftins thus alter the Aß levels in a fashion similar to that described in the brain of AD patients. Aftins therefore constitute new pharmacological tools to investigate this essential aspect of AD, in cell cultures, allowing (1) the detection of inhibitors of Aftin induced action (potential 'anti-AD compounds', including GSIs and GSMs) but also (2) the identification, in the human chemical exposome, of compounds that, like Aftins, might trigger sustained Aß42 production and Aß38 down-regulation (potential 'pro-AD compounds').


Assuntos
Adenina/análogos & derivados , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Modelos Químicos , Fragmentos de Peptídeos/metabolismo , Adenina/farmacologia , Adenina/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Humanos
8.
Toxicol Sci ; 129(2): 332-45, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22987451

RESUMO

Drug-induced liver injury (DILI) in humans is difficult to predict using classical in vitro cytotoxicity screening and regulatory animal studies. This explains why numerous compounds are stopped during clinical trials or withdrawn from the market due to hepatotoxicity. Thus, it is important to improve early prediction of DILI in human. In this study, we hypothesized that this goal could be achieved by investigating drug-induced mitochondrial dysfunction as this toxic effect is a major mechanism of DILI. To this end, we developed a high-throughput screening platform using isolated mouse liver mitochondria. Our broad spectrum multiparametric assay was designed to detect the global mitochondrial membrane permeabilization (swelling), inner membrane permeabilization (transmembrane potential), outer membrane permeabilization (cytochrome c release), and alteration of mitochondrial respiration driven by succinate or malate/glutamate. A pool of 124 chemicals (mainly drugs) was selected, including 87 with documented DILI and 37 without reported clinical hepatotoxicity. Our screening assay revealed an excellent sensitivity for clinical outcome of DILI (94 or 92% depending on cutoff) and a high positive predictive value (89 or 82%). A highly significant relationship between drug-induced mitochondrial toxicity and DILI occurrence in patients was calculated (p < 0.001). Moreover, this multiparametric assay allowed identifying several compounds for which mitochondrial toxicity had never been described before and even helped to clarify mechanisms with some drugs already known to be mitochondriotoxic. Investigation of drug-induced loss of mitochondrial integrity and function with this multiparametric assay should be considered for integration into basic screening processes at early stage to select drug candidates with lower risk of DILI in human. This assay is also a valuable tool for assessing the mitochondrial toxicity profile and investigating the mechanism of action of new compounds and marketed compounds.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/etiologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Animais , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C
9.
PLoS One ; 5(3): e9924, 2010 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-20360986

RESUMO

Current limitations of chemotherapy include toxicity on healthy tissues and multidrug resistance of malignant cells. A number of recent anti-cancer strategies aim at targeting the mitochondrial apoptotic machinery to induce tumor cell death. In this study, we set up protocols to purify functional mitochondria from various human cell lines to analyze the effect of peptidic and xenobiotic compounds described to harbour either Bcl-2 inhibition properties or toxic effects related to mitochondria. Mitochondrial inner and outer membrane permeabilization were systematically investigated in cancer cell mitochondria versus non-cancerous mitochondria. The truncated (t-) Bid protein, synthetic BH3 peptides from Bim and Bak, and the small molecule ABT-737 induced a tumor-specific and OMP-restricted mitochondrio-toxicity, while compounds like HA-14.1, YC-137, Chelerythrine, Gossypol, TW-37 or EM20-25 did not. We found that ABT-737 can induce the Bax-dependent release of apoptotic proteins (cytochrome c, Smac/Diablo and Omi/HtrA2 but not AIF) from various but not all cancer cell mitochondria. Furthermore, ABT-737 addition to isolated cancer cell mitochondria induced oligomerization of Bax and/or Bak monomers already inserted in the mitochondrial membrane. Finally immunoprecipatations indicated that ABT-737 induces Bax, Bak and Bim desequestration from Bcl-2 and Bcl-xL but not from Mcl-1L. This study investigates for the first time the mechanism of action of ABT-737 as a single agent on isolated cancer cell mitochondria. Hence, this method based on MOMP (mitochondrial outer membrane permeabilization) is an interesting screening tool, tailored for identifying Bcl-2 antagonists with selective toxicity profile against cancer cell mitochondria but devoid of toxicity against healthy mitochondria.


Assuntos
Compostos de Bifenilo/farmacologia , Regulação Neoplásica da Expressão Gênica , Membranas Mitocondriais/efeitos dos fármacos , Nitrofenóis/farmacologia , Fragmentos de Peptídeos/química , Proteínas Proto-Oncogênicas/química , Sulfonamidas/farmacologia , Animais , Linhagem Celular Tumoral , Reagentes de Ligações Cruzadas/farmacologia , Feminino , Humanos , Potenciais da Membrana , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/metabolismo , Permeabilidade , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo
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