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1.
Cancer Res ; 79(9): 2244-2256, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30833419

RESUMO

Combined treatment of metastatic melanoma with BRAF and MEK inhibitors has improved survival, but the emergence of resistance represents an important clinical challenge. Targeting ERK is a suitable strategy currently being investigated in melanoma and other cancers. To anticipate possible resistance to ERK inhibitors (ERKi), we used SCH772984 (SCH) as a model ERKi to characterize resistance mechanisms in two BRAF V600E melanoma cell lines. The ERKi-resistant cells were also resistant to vemurafenib (VMF), trametinib (TMT), and combined treatment with either VMF and SCH or TMT and SCH. Resistance to SCH involved stimulation of the IGF1R-MEK5-Erk5 signaling pathway, which counteracted inhibition of Erk1/2 activation and cell growth. Inhibition of IGF1R with linsitinib blocked Erk5 activation in SCH-resistant cells and decreased their growth in 3D spheroid growth assays as well as in NOD scid gamma (NSG) mice. Cells doubly resistant to VMF and TMT or to VMF and SCH also exhibited downregulated Erk1/2 activation linked to stimulation of the IGF1R-MEK5-Erk5 pathway, which accounted for resistance. In addition, we found that the decreased Erk1/2 activation in SCH-resistant cells involved reduced expression and function of TGFα. These data reveal an escape signaling route that melanoma cells use to bypass Erk1/2 blockade during targeted melanoma treatment and offer several possible targets whose disruption may circumvent resistance. SIGNIFICANCE: Activation of the IGF1R-MEK5-Erk5 signaling pathway opposes pharmacologic inhibition of Erk1/2 in melanoma, leading to the reactivation of cell proliferation and acquired resistance.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Indazóis/farmacologia , MAP Quinase Quinase 5/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/patologia , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Piperazinas/farmacologia , Receptor IGF Tipo 1/metabolismo , Animais , Apoptose , Biomarcadores Tumorais , Proliferação de Células , Feminino , Humanos , MAP Quinase Quinase 5/genética , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteína Quinase 7 Ativada por Mitógeno/genética , Receptor IGF Tipo 1/genética , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Food Res Int ; 100(Pt 1): 791-797, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28873751

RESUMO

The aim of the present study was to validate the food safety of CSE, by studying its effect on cytotoxicity (100-20000µg/ml) and genotoxicity (10, 100 and 1000µg/ml) and also to investigate its preventive potential (1, 10 and 100µg/ml) against B(a)P induced DNA damage. Prior to analyses, the antioxidant capacity and the microbiological quality of CSE were tested. DNA damage (strand breaks and oxidized purines/pyrimidines) was evaluated by the alkaline single-cell gel electrophoresis or comet assay. HepG2 cells were pre-treated with CSE (1, 10 and 100µg/ml) for 24h followed by the addition of 100µM B(a)P in presence of CSE for other 24h. Detection of oxidized purines and pyrimidines was carried out using Formamidopyrimidine DNA glycosylase or Endonuclease III enzymes, respectively. Chlorogenic acid (CGA), the major antioxidant present in coffee, was used as a control. Treatment with 100 µM B(a)P significantly increased (p<0.05) levels of DNA strand breaks and oxidized purine and pyrimidine bases. Treatment of HepG2 cells with CSE did not induce either cytotoxicity or genotoxicity. CSE significantly inhibited (p<0.05) genotoxicity induced by B(a)P and the observed effect may be associated to its antioxidant capacity. CGA alone at the concentration present in CSE was effective against B(a)P. Thus, CGA seems to be a contributor to the preventive effect of CSE against B(a)P induced DNA damage in HepG2 cells. In conclusion, CSE presents potential as a natural sustainable chemoprotective agent against the chemical carcinogen B(a)P.


Assuntos
Antioxidantes , Sobrevivência Celular/efeitos dos fármacos , Café/química , Dano ao DNA/efeitos dos fármacos , Extratos Vegetais , Antioxidantes/análise , Antioxidantes/farmacologia , Benzo(a)pireno/toxicidade , Ácido Clorogênico/análise , Ácido Clorogênico/farmacologia , Células Hep G2 , Humanos , Mutagênicos/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/análise , Extratos Vegetais/farmacologia , Reprodutibilidade dos Testes , Sementes/química , Testes de Toxicidade
4.
Int Wound J ; 13(1): 101-9, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24612846

RESUMO

Silver compounds have been used for their medicinal properties for centuries. At present, silver nanoparticles (AgNPs) are reemerging as a viable topical treatment option for infections encountered in burns, open wounds and chronic ulcers. This study evaluated the in vitro mechanisms of two different sizes of AgNPs (4·7 and 42 nm) toxicity in normal human dermal fibroblasts. The toxicity was evaluated by observing cell viability and oxidative stress parameters. In all toxicity endpoints studied (MTT and lactate dehydrogenase assays), AgNPs of 4·7 nm were much more toxic than the large AgNPs (42 nm). The cytotoxicity of both AgNPs was greatly decreased by pre-treatment with the antioxidant N-acetyl-L-cysteine. The oxidative stress parameters showed significant increase in reactive oxygen species levels, depletion of glutathione level and slight, but not statistically significant inactivation of superoxide dismutase, suggesting generation of oxidative stress. Thus, AgNPs should be used with caution for the topical treatment of burns and wounds, medical devices etc, because their toxicity depends on the size, the smaller NPs being much more cytotoxic than the large.


Assuntos
Fibroblastos/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Compostos de Prata/toxicidade , Pele/citologia , Acetilcisteína/farmacologia , Antioxidantes/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Corantes/análise , Glutationa/metabolismo , Humanos , L-Lactato Desidrogenase/análise , Estresse Oxidativo , Tamanho da Partícula , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Sais de Tetrazólio/análise , Tiazóis/análise
5.
Food Chem Toxicol ; 85: 114-9, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26169716

RESUMO

Silver nanoparticles (AgNPs) with antimicrobial activity are by far the most commercialized nano-compound. They are commonly used in medical products and devices, food storage materials, cosmetics and industrial products. Despite the increasing human exposure to AgNPs, they remain a controversial research area with regard to their toxic and genotoxic effects to biological systems. Although previous data have suggested that AgNPs induce toxicity in vitro, the in vivo studies on this topic are very limited. In the present study, the potential genotoxic activity of AgNPs of different sizes (4.7 and 42 nm) was evaluated using the in vivo Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster. Larvae were treated with 25, 30 and 50 µg/ml of AgNPs 4.7 nm, and 250, 500 and 1000 µg/ml of AgNPs 42 nm. Data showed that AgNPs at the applied concentrations did not modify the spontaneous frequencies of spots indicating lack of mutagenic and recombinogenic activity. However, both AgNPs induced pigmentation defects and reduction in locomotor ability in adult flies. Therefore, further experiments must be carried out to gain a better understanding of the mechanism of action of AgNPs to ensure their safe use.


Assuntos
Drosophila melanogaster/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Nanopartículas Metálicas/toxicidade , Mutagênicos/toxicidade , Mutação/efeitos dos fármacos , Recombinação Genética/efeitos dos fármacos , Prata/toxicidade , Administração Oral , Animais , Animais Geneticamente Modificados , Anti-Infecciosos/administração & dosagem , Anti-Infecciosos/química , Anti-Infecciosos/toxicidade , Cruzamentos Genéticos , Relação Dose-Resposta a Droga , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Poluentes Ambientais/administração & dosagem , Poluentes Ambientais/química , Larva/efeitos dos fármacos , Larva/genética , Larva/crescimento & desenvolvimento , Locomoção/efeitos dos fármacos , Nanopartículas Metálicas/administração & dosagem , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Microscopia Eletrônica de Transmissão , Testes de Mutagenicidade , Mutagênicos/administração & dosagem , Mutagênicos/química , Tamanho da Partícula , Pigmentação/efeitos dos fármacos , Prata/administração & dosagem , Prata/química , Asas de Animais
6.
Toxicol Mech Methods ; 25(4): 287-95, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25798650

RESUMO

Silver and gold nanoparticles (Ag-AuNPs) are currently some of the most manufactured nanomaterials. Accordingly, the hazards associated with human exposure to Ag-AuNPs should be investigated to facilitate the risk assessment process. In particular, because pulmonary exposure to Ag-AuNPs occurs during handling of these nanoparticles, it is necessary to evaluate the toxic response in pulmonary cells. The aim of this study was to evaluate the in vitro mechanisms of toxicity of different sizes of silver (4.7 and 42 nm) and gold nanoparticles (30, 50 and 90 nm) in human pulmonary fibroblasts (HPF). The toxicity was evaluated by observing cell viability and oxidative stress parameters. Data showed that AgNPs-induced cytotoxicity was size-dependent, whereas the AuNPs of the three sizes showed similar cytotoxicity. Silver nanoparticles of 4.7 nm were much more toxic than the large silver nanoparticles and the AuNPs. However, the pre-treatment with the antioxidant, N-acetyl-L-cysteine, protected HPF cells against treatment with Ag-AuNPs. The oxidative stress parameters revealed significant increase in reactive oxygen species levels, depletion of glutathione level and slight, but not statistically significant inactivation of superoxide dismutase, suggesting generation of oxidative stress. Hence, care has to be taken while processing and formulating the Ag-AuNPs till their final finished product.


Assuntos
Fibroblastos/efeitos dos fármacos , Ouro/toxicidade , Nanopartículas Metálicas/toxicidade , Prata/toxicidade , Acetilcisteína/farmacologia , Antioxidantes/farmacologia , Cardiotônicos/farmacologia , Células Cultivadas , Humanos , Pulmão/citologia , Estresse Oxidativo/efeitos dos fármacos , Tamanho da Partícula , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Sais de Tetrazólio/metabolismo , Tiazóis/metabolismo
7.
J Appl Toxicol ; 34(4): 413-23, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24243578

RESUMO

Silver nanoparticles (AgNPs), which have well-known antimicrobial properties, are extensively used in various medical and general applications. In spite of the widespread use of AgNPs, relatively few studies have been undertaken to determine the cytotoxic effects of AgNPs. The aim of this study was investigate how AgNPs of different sizes (4.7 and 42 nm) interact with two different tumoral human cell lines (hepatoma [HepG2] and leukemia [HL-60]). In addition, glutathione depletion, inhibition of superoxide dismutase (SOD) and reactive oxygen species (ROS) generation were used to evaluate feasible mechanisms by which AgNPs exerted its toxicity. AgNPs of 4.7 nm and 42 nm exhibited a dramatic difference in cytotoxicity. Small AgNPs were much more cytotoxic than large AgNPs. A difference in the cellular response to AgNPs was found. HepG2 cells showed a higher sensitivity to the AgNPs than HL-60. However, the cytotoxicity induced by AgNPs was efficiently prevented by NAC treatment, which suggests that oxidative stress is primarily responsible for the cytotoxicity of AgNPs. Furthermore, cellular antioxidant status was disturbed: AgNPs exposure caused ROS production, glutathione depletion and slight, but not statistically significant inactivation of SOD.


Assuntos
Nanopartículas Metálicas/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Prata/toxicidade , Acetilcisteína/farmacologia , Técnicas de Cultura de Células , Sobrevivência Celular/efeitos dos fármacos , Depuradores de Radicais Livres/farmacologia , Glutationa/metabolismo , Células HL-60 , Células Hep G2 , Humanos , Nanopartículas Metálicas/química , Microscopia Eletrônica de Transmissão , Tamanho da Partícula , Prata/química , Superóxido Dismutase/metabolismo , Propriedades de Superfície
8.
Toxicol Mech Methods ; 24(3): 161-72, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24274460

RESUMO

Due to their exceptional properties, gold nanoparticles (AuNPs) have shown promising medical and technological applications in the treatment of cancer and the development of antimicrobial packaging and time-temperature indicators in the food sector. However, little is known about their cytotoxicity when they come into contact with biological systems. The aim of this work was to compare the effects of three commercially available AuNPs of different sizes (30, 50 and 90 nm) on human leukemia (HL-60) and hepatoma (HepG2) cell lines. AuNP-induced cytotoxicity was dose and time-dependent, with IC50 values higher than 15 µg/mL. Nanoparticle (NP) size and cell line slightly influenced on the cytotoxicity of AuNPs, although HL-60 cells proved to be more sensitive to the cytotoxic response than HepG2. N-Acetyl-L-cysteine (NAC) protected HL-60 and HepG2 cells only against treatment with 30 nm AuNPs. In both cell types, glutathione (GSH) content was drastically depleted after 72 h of incubation with the three AuNPs (less than 30% in all cases), while the reduction of superoxide dismutase activity (SOD) activity depended on cell line. HepG2, but not HL-60 cells, exhibited a decrease of SOD activity (∼ 45% of activity). The three AuNPs also caused a two-fold elevation of reactive oxygen species (ROS) production in both cell lines. Thus, protective effect of NAC, depletion of GSH and increase of ROS appear to be determined by NP size and indicate that oxidative stress contributes to cytotoxicity of AuNPs.


Assuntos
Ouro/farmacologia , Nanopartículas Metálicas/administração & dosagem , Estresse Oxidativo , Acetilcisteína/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Glutationa/análise , Humanos , Tamanho da Partícula , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo
9.
Acta toxicol. argent ; 21(2): 102-109, dic. 2013. ilus
Artigo em Espanhol | BINACIS | ID: bin-130342

RESUMO

En los últimos años, la evolución en el desarrollo de productos elaborados a partir de nanotecnología ha experimentado un espectacular crecimiento. En particular, las nanopartículas de oro han despertado gran interés en los sectores biomédico y alimentario, donde se ha descrito su utilización en el tratamiento frente al cáncer o como parte integrante de envases resistentes a la abrasión, con propiedades antimicrobianas. Por tanto, se cree que la exposición humana a las nanopartículas de oro aumentará considerablemente en los próximos años, pudiendo tener esto repercusiones sobre la salud. En este marco, el estudio de la toxicología de las nanopartículas ha revelado que su toxicidad depende de multitud de factores. Además, en la bibliografía hay cierta controversia en torno a los posibles efectos citotóxicos inducidos por las nanopartículas de oro. Diversos estudios de exposición in vitro han destacado su inocuidad en algunas líneas celulares, mientras que otros trabajos demostraron respuesta citotóxica. La siguiente revisión tiene por objeto describir las propiedades más relevantes de las nanopartículas de oro considerando sus potenciales aplicaciones en medicina y en la industria de los alimentos, así como examinar su posible toxicidad, con especial énfasis en los estudios de citotoxicidad in vitro disponibles hasta el momento.(AU)


In the recent years, the development of nanotechnology-based products has experienced a spectacular growth. Especially, gold nanoparticles have awoken a great interest in the biomedical and food sector, where their applications in cancer treatment as well as their incorporation in abrasion resistant and antimicrobial packaging have been described. Therefore, it is believed that human exposure to gold nanoparticles will increase considerably in the next few years, which may arise possible human health hazards. Hence, toxicology studies on nanoparticles revealed that their toxicity depends on various factors. Furthermore, there is some controversy regarding to gold nanoparticle-induced cytotoxicity. Several in vitro studies have reported that gold nanoparticles are innocuous, while some investigations have demonstrated a cytotoxic response after the exposure to these. The aim of this review is to describe the most relevant properties of gold nanoparticles according to their possible applications in medicine and in food industry, as well as to provide information about their possible toxic effects, taking into account the cytotoxic in vitro studies published at present.(AU)

10.
Acta toxicol. argent ; 21(2): 102-109, dic. 2013. ilus
Artigo em Espanhol | LILACS | ID: lil-708420

RESUMO

En los últimos años, la evolución en el desarrollo de productos elaborados a partir de nanotecnología ha experimentado un espectacular crecimiento. En particular, las nanopartículas de oro han despertado gran interés en los sectores biomédico y alimentario, donde se ha descrito su utilización en el tratamiento frente al cáncer o como parte integrante de envases resistentes a la abrasión, con propiedades antimicrobianas. Por tanto, se cree que la exposición humana a las nanopartículas de oro aumentará considerablemente en los próximos años, pudiendo tener esto repercusiones sobre la salud. En este marco, el estudio de la toxicología de las nanopartículas ha revelado que su toxicidad depende de multitud de factores. Además, en la bibliografía hay cierta controversia en torno a los posibles efectos citotóxicos inducidos por las nanopartículas de oro. Diversos estudios de exposición in vitro han destacado su inocuidad en algunas líneas celulares, mientras que otros trabajos demostraron respuesta citotóxica. La siguiente revisión tiene por objeto describir las propiedades más relevantes de las nanopartículas de oro considerando sus potenciales aplicaciones en medicina y en la industria de los alimentos, así como examinar su posible toxicidad, con especial énfasis en los estudios de citotoxicidad in vitro disponibles hasta el momento.


In the recent years, the development of nanotechnology-based products has experienced a spectacular growth. Especially, gold nanoparticles have awoken a great interest in the biomedical and food sector, where their applications in cancer treatment as well as their incorporation in abrasion resistant and antimicrobial packaging have been described. Therefore, it is believed that human exposure to gold nanoparticles will increase considerably in the next few years, which may arise possible human health hazards. Hence, toxicology studies on nanoparticles revealed that their toxicity depends on various factors. Furthermore, there is some controversy regarding to gold nanoparticle-induced cytotoxicity. Several in vitro studies have reported that gold nanoparticles are innocuous, while some investigations have demonstrated a cytotoxic response after the exposure to these. The aim of this review is to describe the most relevant properties of gold nanoparticles according to their possible applications in medicine and in food industry, as well as to provide information about their possible toxic effects, taking into account the cytotoxic in vitro studies published at present.


Assuntos
Nanopartículas Metálicas/toxicidade , Ouro/toxicidade , Citotoxinas , Nanotecnologia/legislação & jurisprudência , Nanopartículas Metálicas/análise , Nanopartículas Metálicas/uso terapêutico , Ouro/uso terapêutico
11.
Pharmacogn Mag ; 9(35): 231-7, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23930007

RESUMO

BACKGROUND: Current evidence supports that consumption of polyphenols has beneficial effects against numerous diseases mostly associated with their antioxidant activity. Honey is a good source of antioxidants since it contains a great variety of phenolic compounds. OBJECTIVE: The main objective of this work was to investigate the antiproliferative and apoptotic effects of three crude commercial honeys of different floral origin (heather, rosemary and polyfloral honey) from Madrid Autonomic Community (Spain) as well as of an artificial honey in human peripheral blood promyelocytic leukemia cells (HL-60). MATERIAL AND METHODS: HL-60 cells were cultured in the presence of honeys at various concentrations for up to 72 hours and the percentage of cell viability was evaluated by MTT assay. Apoptotic cells were identified by chromatin condensation and flow cytometry analysis. ROS production was determined using 2´,7´-dichlorodihydrofluorescein diacetate (H2DCFDA). RESULTS: The three types of crude commercial honey induced apoptosis in a concentration and time dependent-manner. In addition, honeys with the higher phenolic content, heather and polyfloral, were the most effective to induce apoptosis in HL-60 cells. However, honeys did not generate reactive oxygen species (ROS) and N-acetyl-L-cysteine (NAC) could not block honeys-induced apoptosis in HL-60 cells. CONCLUSION: These data support that honeys induced apoptosis in HL-60 cells through a ROS-independent cell death pathway. Moreover, our findings indicate that the antiproliferative and apoptotic effects of honey varied according to the floral origin and the phenolic content.

12.
J Sci Food Agric ; 93(12): 2995-3000, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23504582

RESUMO

BACKGROUND: Honey contains a variety of polyphenols and represents a good source of antioxidants, while the human diet often contains compounds that can cause DNA damage. The present study investigated the protective effect of three commercial honey samples of different floral origin (rosemary, heather and heterofloral) from Madrid Autonomic Community (Spain) as well as an artificial honey on DNA damage induced by dietary mutagens, using a human hepatoma cell line (HepG2) as in vitro model system and evaluation by the alkaline single-cell gel electrophoresis or comet assay. RESULTS: Rosemary, heather and heterofloral honeys protected against DNA strand breaks induced by N-nitrosopyrrolidine (NPYR), benzo(a)pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), but none of the honey samples tested prevented DNA strand breaks induced by N-nitrosodimethylamine (NDMA). Heterofloral and heather (unifloral) honeys with higher phenolic content were most effective in protecting HepG2 cells against DNA damage induced by food mutagens. Heterofloral honey was more protective against NPYR and BaP, while heather honey was more protective against PhIP. Artificial honey did not show a protective effect against DNA damage induced by any of the food mutagens tested, indicating that the protective effects of honeys could not be due to their sugar components. CONCLUSION: The results suggest that the protective effect of three kinds of Spanish honey of different floral origin could be attributed in part to the phenolics present in the samples. Honeys with higher phenolic content, i.e. heather and heterofloral honeys, were most effective in protecting against food mutagen-induced DNA damage in HepG2 cells. In addition, a possible synergistic effect between other minor honey components could also be involved.


Assuntos
Anticarcinógenos/metabolismo , Dano ao DNA , Contaminação de Alimentos , Alimento Funcional , Mel , Neoplasias Hepáticas/prevenção & controle , Mutagênicos/toxicidade , Benzo(a)pireno/antagonistas & inibidores , Benzo(a)pireno/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Ensaio Cometa , Quebras de DNA/efeitos dos fármacos , Ericaceae/metabolismo , Alimento Funcional/análise , Células Hep G2 , Mel/análise , Humanos , Imidazóis/antagonistas & inibidores , Imidazóis/toxicidade , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/metabolismo , Modelos Biológicos , Mutagênicos/química , N-Nitrosopirrolidina/química , N-Nitrosopirrolidina/toxicidade , Fenóis/análise , Fenóis/metabolismo , Rosmarinus/metabolismo , Espanha
13.
Toxicol Mech Methods ; 23(3): 153-60, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23278213

RESUMO

Silver nanoparticles (AgNPs), which have well-known antimicrobial properties, are extensively used in various medical and general applications. Despite the widespread use of AgNPs, relatively few studies have been undertaken to determine the toxicity effects of AgNPs exposure. The aim of the present work was to study how AgNPs interact with four different human cell lines (hepatoma, leukemia, dermal and pulmonary fibroblast) in order to understand the impact of such nanomaterials on cellular biological functions. For toxicity evaluations, mitochondrial function (MTT assay) and membrane leakage of lactate dehydrogenase (LDH assay) were assessed under control and exposed conditions (24, 48 and 72 h of exposure). Furthermore, we evaluated the protective effect of N-acetyl-l-cysteine (NAC) against AgNP-induced cytotoxicity. Results showed that mitochondrial function decreased in all cell lines exposed to AgNPs at 6.72-13.45 µg/ml. LDH leakage also increased in all cell lines exposed to AgNPs (6.72-13.45 µg/ml). However, the cytotoxic effect of AgNPs (13.45 µg/ml) was prevented by pretreatment of different concentrations of NAC (1-20 mM). Our findings indicate that AgNPs are cytotoxic on human tumor and normal cells, the tumor cells being more sensitive to the cytotoxic effect of AgNPs. In addition, NAC protects human cells from cytotoxicity of AgNPs, suggesting that oxidative stress is in part responsible of this effect.


Assuntos
Antineoplásicos/farmacologia , Fibroblastos/efeitos dos fármacos , Nanopartículas Metálicas , Neoplasias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Compostos de Prata/farmacologia , Acetilcisteína/farmacologia , Antineoplásicos/toxicidade , Antioxidantes/farmacologia , Biomarcadores/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Citoproteção , Relação Dose-Resposta a Droga , Fibroblastos/metabolismo , Fibroblastos/patologia , Células HL-60 , Células Hep G2 , Humanos , L-Lactato Desidrogenase/metabolismo , Nanopartículas Metálicas/toxicidade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neoplasias/patologia , Compostos de Prata/toxicidade , Fatores de Tempo
14.
J Appl Toxicol ; 32(12): 986-93, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21935971

RESUMO

Numerous studies have shown the potential of dietary polyphenols as anticarcinogenic agents. The aim of the present study was to evaluate the apoptotic effects of piceatannol and myricetin, naturally occurring polyphenols in red wine, alone or in combination, in two human cell lines: HL-60 (leukemia) and HepG2 (hepatoma). Apoptotic cells were identified by chromatin condensation, poly(ADP-ribose) polymerase cleavage and flow cytometry analysis. Results from TUNEL assay showed that piceatannol or myricetin alone induced apoptotic cell death in a concentration- and time-dependent manners in HL-60 cells. Furthermore, in combined treatment the percentage of apoptotic HL-60 cells was significantly higher. Nevertheless, the percentage of TUNEL positive HepG2 cells only was significant after piceatannol treatment and in combined treatment was even lower than in cells treated with piceatannol alone. Moreover, we also studied the relative reactive oxygen species (ROS) production. Our results indicate that apoptosis induced by piceatannol or myricetin occurs through an ROS-independent cell death pathway. In conclusion, piceatannol and myricetin synergistically induced apoptosis in HL-60 cells but not in HepG2 cells. These findings suggest that the potential anticarcinogenic properties of dietary polyphenols depend largely on the cell line used. The relevance of these data needs to be verified in human epidemiological studies.


Assuntos
Anticarcinógenos/farmacologia , Apoptose/efeitos dos fármacos , Flavonoides/farmacologia , Polifenóis/farmacologia , Estilbenos/farmacologia , Anticarcinógenos/administração & dosagem , Western Blotting , Técnicas de Cultura de Células , Sobrevivência Celular/efeitos dos fármacos , Flavonoides/administração & dosagem , Citometria de Fluxo , Células HL-60 , Células Hep G2 , Humanos , Marcação In Situ das Extremidades Cortadas , Polifenóis/administração & dosagem , Espécies Reativas de Oxigênio/metabolismo , Estilbenos/administração & dosagem
15.
J Appl Toxicol ; 31(1): 53-62, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20583320

RESUMO

The aim of the present study was to evaluate the protective effect of (+)-catechin and (-)-epicatechin against 2-amino-3,8- dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]-quinoxaline (4,8-diMeIQx) and 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP)-induced DNA damage in human hepatoma cells (HepG2). DNA damage (strand breaks and oxidized purines/pyrimidines) was evaluated by the alkaline single-cell gel electrophoresis or comet assay. Increasing concentrations of 8-MeIQx, 4,8-diMeIQx and PhIP induced a significant increase in DNA strand breaks and oxidized purines and pyrimidines in a dose-dependent manner. Among those, PhIP (300 µm) exerted the highest genotoxicity. (+)-Catechin exerted protection against oxidized purines induced by 8-MeIQx, 4,8-diMeIQx and PhIP. Oxidized pyrimidines and DNA strand breaks induced by PhIP were also prevented by (+)-catechin. Otherwise, (-)-epicatechin protected against the oxidized pyrimidines induced by PhIP and the oxidized purines induced by 8-MeIQx and 4,8-diMeIQx. One feasible mechanism by which (+)-catechin and (-)-epicatechin exert their protective effect towards heterocyclic amines-induced oxidative DNA damage may be by modulation of phase I and II enzyme activities. The ethoxyresorufin O-deethylation (CYP1A1) activity was moderately inhibited by (+)-catechin, while little effect was observed by (-)-epicatechin. However, (+)-catechin showed the greatest increase in UDP-glucuronyltransferase activity. In conclusion, our results clearly indicate that (+)-catechin was more efficient than (-)-epicatechin in preventing DNA damage (strand breaks and oxidized purines/pyrimidines) induced by PhIP than that induced by 8-MeIQx and 4,8-diMeIQx.


Assuntos
Carcinoma Hepatocelular/metabolismo , Catequina/química , Catequina/farmacologia , Dano ao DNA/efeitos dos fármacos , Imidazóis/toxicidade , Quinoxalinas/toxicidade , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Ensaio Cometa , Relação Dose-Resposta a Droga , Células Hep G2 , Humanos , Oxirredução
16.
Cell Biol Int ; 33(12): 1280-6, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19748591

RESUMO

The human hepatoma cell line (HepG2) exhibited a dose and time-dependent apoptotic response following treatment with N-Nitrosopiperidine (NPIP) and N-Nitrosodibutylamine (NDBA), two recognized human carcinogens. Our results showed a significant apoptotic cell death (95%) after 24h treatment with NDBA (3.5 mM), whereas it was necessary to use high doses of NPIP (45 mM) to obtain a similar percentage of apoptotic cells (86%). In addition, both extrinsic (caspase-8) and intrinsic pathway (caspase-9) could be implicated in the N-Nitrosamines-induced apoptosis. This study also addresses the role of reactive oxygen species (ROS) as intermediates for apoptosis signaling. A significant increase in ROS levels was observed after NPIP treatment, whereas NDBA did not induce ROS. However, N-acetylcysteine (NAC) did not block NPIP-induced apoptosis. All these findings suggest that NPIP and NDBA induce apoptosis in HepG2 cells via a pathway that involves caspases but not ROS.


Assuntos
Alquilantes/farmacologia , Apoptose/efeitos dos fármacos , Carcinógenos/farmacologia , Caspases/metabolismo , Dietilnitrosamina/farmacologia , Nitrosaminas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Células Hep G2 , Humanos , Marcação In Situ das Extremidades Cortadas , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Espécies Reativas de Oxigênio/metabolismo
17.
J Appl Toxicol ; 29(5): 403-13, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19301245

RESUMO

The aim of this work was to determine the effect of vitamin C, diallyl disulfide (DADS) and dipropyl disulfide (DPDS) towards N-nitrosopiperidine (NPIP) and N-nitrosodibutylamine (NDBA)-induced apoptosis in human leukemia (HL-60) and hepatoma (HepG2) cell lines using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. None of the vitamin C (5-50 microm), DADS and DPDS (1-5 microm) concentrations selected induced a significant percentage of apoptosis. In simultaneous treatments, vitamin C, DADS and DPDS reduced the apoptosis induced by NPIP and NDBA in HL-60 and HepG2 cells (around 70% of reduction). We also investigated its scavenging activities towards reactive oxygen species (ROS) produced by NPIP and NDBA using 2',7'-dichlorodihydrofluorescein diacetate in both cell lines. ROS production induced by both N-nitrosamine was reduced to control levels by vitamin C (5-50 microm) in a dose-dependent manner. However, DADS (5 microm) increased ROS levels induced by NPIP and NDBA in HL-60 (40 and 20% increase, respectively) and HepG2 cells (18% increase), whereas DPDS was more efficient scavenger of ROS at the lowest concentration (1 microm) in both HL-60 (52 and 25% reduction, respectively) and HepG2 cells (24% reduction). The data demonstrated that the scavenging ability of vitamin C and DPDS could contribute to inhibition of the NPIP- and NDBA-induced apoptosis. However, more than one mechanism, such as inhibition of phase I and/or induction of phase II enzymes, could be implicated in the protective effect of dietary antioxidants towards NPIP- and NDBA-induced apoptosis in HL-60 and HepG2 cells.


Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Suplementos Nutricionais , Nitrosaminas/toxicidade , Compostos Alílicos/administração & dosagem , Compostos Alílicos/farmacologia , Antioxidantes/administração & dosagem , Ácido Ascórbico/administração & dosagem , Ácido Ascórbico/farmacologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Técnicas de Cultura de Células , Dissulfetos/administração & dosagem , Dissulfetos/farmacologia , Relação Dose-Resposta a Droga , Células HL-60 , Humanos , Marcação In Situ das Extremidades Cortadas , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
18.
Eur J Nutr ; 47(8): 479-90, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18974940

RESUMO

BACKGROUND: Dietary polyphenols have been reported to have a variety of biological actions, including anticarcinogenic and antioxidant activities. AIM OF THE STUDY: In the present study we investigated the protective effect of dietary polyphenols against N-nitrosodimethylamine (NDMA), N-nitrosopyrrolidine (NPYR) and benzo(a)pyrene (BaP)-induced DNA damage (strand breaks and oxidized purines/pyrimidines) in HepG2 cells. METHODS: Human hepatocellular carcinoma (HepG2) cells, which retain many specialized liver functions and drug metabolizing enzyme activities, were used as in vitro model for human hepatocytes. NDMA, NPYR and BaP were employed to induce DNA damage. DNA damage (strand breaks, oxidized pyrimidines and oxidized purines) was evaluated by the alkaline single cell gel electrophoresis or comet assay. RESULTS: None of the polyphenols concentrations tested in presence or absence of Fpg (formamidopyrimidine-DNA glycosylase), or Endo III (Endonuclease III) caused DNA damage per se. Increasing concentrations of BaP (25-100 microM) induced a significant increase of DNA strand breaks, Fpg and Endo III sensitive sites in a dose dependent manner. Myricetin and quercetin decreased DNA strand breaks and oxidized pyrimidines induced by NDMA, but not oxidized purines. However, both flavonoids reduced oxidized pyrimidines and purines induced by NPYR. DNA strand breaks induced by NPYR were prevented by quercetin, but not by myricetin. BaP-induced DNA strand breaks and oxidized pyrimidines were strongly reduced by myricetin and quercetin, respectively. While oxidized purines induced by BaP were reduced by quercetin, myricetin had no protective effect. (+)-Catechin and (-)-epicatechin reduced DNA strand breaks, oxidized pyrimidines and oxidized purines induced by NDMA. DNA strand breaks, and oxidized purines induced by NPYR were also prevented by (+)-catechin and (-)-epicatechin, while the maximum reduction of oxidized pyrimidines was found by (+)-catechin and (-)-epicatechin at 10 microM. (+)-Catechin and (-)-epicatechin decreased also DNA strand breaks and oxidized pyrimidines but not oxidized purines induced by BaP. CONCLUSIONS: Our results clearly indicate that polyphenols protect human derived cells against DNA strand breaks and oxidative DNA damage effects of NDMA, NPYR or BaP, three carcinogenic compounds which occur in the environment.


Assuntos
Anticarcinógenos/farmacologia , Benzo(a)pireno/toxicidade , Dano ao DNA/efeitos dos fármacos , Flavonoides/farmacologia , Nitrosaminas/toxicidade , Fenóis/farmacologia , Anticarcinógenos/administração & dosagem , Carcinógenos/toxicidade , Linhagem Celular Tumoral , Ensaio Cometa , DNA/efeitos dos fármacos , DNA/metabolismo , Relação Dose-Resposta a Droga , Flavonoides/administração & dosagem , Humanos , Fenóis/administração & dosagem , Polifenóis
19.
J Appl Toxicol ; 28(6): 788-96, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18344201

RESUMO

The aim of this study was to evaluate the effect of vitamin C towards N-nitrosopyrrolidine (NPYR)- and N-nitrosodimethylamine (NDMA)-induced apoptosis in human hepatoma (HepG2) and leukemia (HL-60) cell lines using flow cytometry analysis and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay (TUNEL). None of the vitamin C concentrations tested (1-100 microM) caused cytotoxicity in HepG2 cells. However, there were significant losses of HL-60 cells viability, measured by MTT assay, 72 h after treatment with 50 and 100 microM vitamin C (29 and 46%, respectively). Moreover, an increase of lactate dehydrogenase release was significant with 50 microM at 72 h (28%) and with 100 microM of vitamin C at 48 and 72 h (27 and 36%, respectively). Also, the percentage of apoptotic HL-60 cells found in TUNEL assay increased to 21% when they were treated with 100 microM vitamin C for 72 h. Thus, in subsequent simultaneous treatments with NPYR (30 and 50 mM) or NDMA (27 and 68 mM) and vitamin C, concentrations of 5-50 microM vitamin C were used. Our results revealed that vitamin C, at all concentrations and times tested, reduced the apoptosis induced by NPYR and NDMA in both cell lines, showing a similar effect in HepG2 and HL-60 cells towards NPYR (50 mM)--65 and 63% of reduction, respectively--whereas towards NDMA (27 mM) the inhibition was higher in HL-60 than in HepG2 cells--75 and 57%, respectively. Therefore, our findings suggest that inhibition of apoptosis may be one of the mechanisms by which vitamin C exerts its protective effect.


Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Ácido Ascórbico/farmacologia , Nitrosaminas/antagonistas & inibidores , Nitrosaminas/toxicidade , Carcinógenos/toxicidade , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/metabolismo , Agonistas de Aminoácidos Excitatórios/toxicidade , Células HL-60 , Humanos , Marcação In Situ das Extremidades Cortadas , L-Lactato Desidrogenase/metabolismo , Neoplasias Hepáticas/patologia , N-Metilaspartato/antagonistas & inibidores , N-Metilaspartato/toxicidade , N-Nitrosopirrolidina/toxicidade , Sais de Tetrazólio , Tiazóis
20.
J Appl Toxicol ; 28(4): 455-65, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-17929238

RESUMO

N-nitrosopiperidine (NPIP) and N-nitrosodibutylamine (NDBA) belong to a group of N-nitrosamines that are widely distributed in foodstuffs and the occupational environment. In the present study, the human promyelocytic leukemia cell line HL-60, was used to characterize the apoptotic effects of N-nitrosamines, and to examine the production of reactive oxygen species (ROS). Apoptotic cells were identified by (i) chromatin condensation (ii) flow cytometry analysis and (iii) poly(ADP-ribose) polymerase (PARP) cleavage. NPIP and NDBA induced morphological changes consistent with apoptotic events in HL-60 cells. Flow cytometry analysis showed that both N-nitrosamines induced apoptotic cell death in a concentration and time dependent-manner. It was observed that NDBA was stronger than NPIP, since it induced a significant apoptotic cell death after 18 h starting from a concentration of 2 mm, whereas NPIP was effective at 10 mm. Furthermore, PARP was markedly cleaved with 0.5 mm of NDBA and 5 mm of NPIP after treatments for 3 and 18 h, respectively. Finally, the ROS level was found to be elevated after 0.5 h of treatment with both N-nitrosamines. Antioxidant N-acetylcysteine (NAC) completely inhibited the ROS production induced by NPIP and NDBA. However, this action seems not to be associated with the apoptosis because NAC did not block N-nitrosamines-induced apoptosis. The data demonstrate that NPIP and NDBA induce apoptosis and ROS production in HL-60 cells.


Assuntos
Apoptose/efeitos dos fármacos , Carcinógenos/toxicidade , Leucemia/patologia , Nitrosaminas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/farmacologia , Antioxidantes/farmacologia , Western Blotting , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Relação Dose-Resposta a Droga , Citometria de Fluxo , Células HL-60 , Humanos , Marcação In Situ das Extremidades Cortadas , Leucemia/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Fatores de Tempo
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