RESUMEN
The purpose of the study was to characterize the involvement of reactive oxygen species (ROS) in mediating the cytotoxic effects of arsenic trioxide (ATO) in combination with sulindac or its metabolites: sulfide (SS) and sulfone (SF) on human leukemic cell lines. Jurkat, HL-60, K562, and HPB-ALL cells were exposed to the drugs alone or in combinations. Cell viability was measured using WST-1 or XTT reduction tests and ROS production by dichlorodihydrofluorescein diacetate staining (flow cytometry). Modulation of (a) intracellular glutathione (GSH) level was done by using L: -buthionine sulfoximine (BSO) or diethylmaleate (DEM), (b) NADPH oxidase by using diphenyleneiodonium (DPI), and (c) MAP kinases by using SB202190 (p38), SP600125 (JNK), and U0126 (ERK) inhibitors. ATO cytotoxicity (0.5 or 1 µM) was enhanced by sulindacs, with higher activity showed by the metabolites. Strong cytotoxic effects appeared at SS and SF concentrations starting from 50 µM. The induction of ROS production seemed not to be the major mechanism responsible for the cytotoxicity of the combinations. A strong potentiating effect of BSO on ATO cytotoxicity was demonstrated; DEM (10-300 µM) and DPI (0.0025-0.1 µM; 72 h) did not influence the effects of ATO. Some significant decreases in the viability of the cells exposed to ATO in the presence of MAPK inhibitors comparing with the cells exposed to ATO alone were observed; however, the effects likely resulted from a simple additive cytotoxicity of the drugs. The combinations of ATO with sulindacs offer potential therapeutic usefulness.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Leucemia/patología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Estrés Oxidativo/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Trióxido de Arsénico , Arsenicales/administración & dosificación , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Glutatión/metabolismo , Humanos , Leucemia/tratamiento farmacológico , Leucemia/metabolismo , Óxidos/administración & dosificación , Especies Reactivas de Oxígeno/metabolismo , Sulindac/administración & dosificación , Sulindac/análogos & derivadosRESUMEN
This study was undertaken to investigate the genotoxic interactions between the common environmental pollutants: arsenic (As), cadmium (Cd) and benzo(a)pyrene (BaP), which are known to be human carcinogens. C57BL/6J/Han mice were pre-treated with 100mg cadmium chloride (Cd(2+))/L or 50mg sodium arsenite (As(3+))/L in drinking water for 7 days and then given a single dose of 200mg BaP/kg bw by intra-peritoneal injection. A third group of mice did not receive the pre-treatment and was given BaP alone. Mice were sacrificed before or at 12, 24, 48 or 72h after BaP administration. Chromosome damage in bone-marrow cells was assessed by use of the micronucleus test. The study revealed that BaP induced a statistically significant increase in micronucleus (MN) frequency at 48h after administration. In animals exposed to Cd in drinking water no enhancement of genotoxicity was observed compared with the control group that was given tap water only. In Cd/BaP co-exposed animals, the MN frequency at respective time points did not differ from that for the animals exposed solely to BaP. A statistically higher MN frequency was found in bone marrow of animals exposed to As compared with controls that received tap water (0.92+/-0.29% versus 0.38+/-0.13%, respectively). This effect was even more pronounced after combined exposure to As and BaP. In the co-exposed animals, significantly elevated levels of MN were detected in samples examined at 12, 24 and 48h after BaP administration, compared with animals receiving BaP alone (1.14+/-0.31%, 1.26+/-0.3% and 2.02+/-0.45% versus 0.44+/-0.13%, 0.44+/-0.11% and 1.04+/-0.44%, respectively). These findings imply strong interactions between As and BaP, but not between Cd and BaP, in inducing DNA damage in polychromatic erythrocytes in mouse bone-marrow.
Asunto(s)
Arsénico/farmacología , Benzo(a)pireno/toxicidad , Médula Ósea/efectos de los fármacos , Cadmio/farmacología , Micronúcleos con Defecto Cromosómico/inducido químicamente , Animales , Arsénico/toxicidad , Cadmio/toxicidad , Interacciones Farmacológicas , Masculino , Ratones , Ratones Endogámicos C57BL , Pruebas de Micronúcleos , Contaminantes Químicos del Agua/toxicidadRESUMEN
Existing data indicate that the increase of il-1beta gene expression can be a promising marker of Langerhans cells activation after exposure to contact sensitizers. In this study, we were interested in development of an alternative in vitro screening test detecting such sensitizers. Two IL-1beta reporter constructs containing the enhanced green fluorescent protein (GFP) gene and mouse IL-1beta promoter fragments of varying lengths (-500 bp and -4093 bp) were used for transient transfections of J771A.1 murine monocyte-macrophage cells. As a result of the transfections performed using Lipofectamine reagent we did not observe any GFP fluorescence after stimulation of the cells with LPS as well as known sensitizers (potassium tetrachloroplatinate, dinitrochlorobenzene and nickel sulfate). Low transfection efficiency of J774A.1 cells (less than 0.1%) was confirmed using control plasmid containing GFP gene under the control of cytomegalovirus promoter. The fact that, using the same conditions, we were able to transfect murine fibroblasts 3T3-L1 with the control plasmid very efficiently, may support the theory of high metabolic activity of macrophages being responsible for the extremely low transfection efficiency. These data suggest limited suitability of J774A.1 cell line for transient transfections using cationic liposomes.
Asunto(s)
Interleucina-1/genética , Macrófagos , Pruebas de Toxicidad/métodos , Transfección/métodos , Células 3T3-L1 , Alérgenos/toxicidad , Animales , Línea Celular , ADN/metabolismo , Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Interleucina-1/metabolismo , Irritantes/toxicidad , Lípidos , Liposomas , Ratones , Regiones Promotoras Genéticas , ARN Mensajero/metabolismoRESUMEN
It has been postulated that exposure to nitrous oxide and halogenated anaesthetics is associated with various adverse health effects such as neurological and reproductive abnormalities or impairment of hepatic functions. In spite of the quite well known genotoxic effects of exposure to nitrous oxide in vivo, the mechanisms of these effects are still not clear. The aim of this study was to assess the frequency of micronuclei and to identify the type of chromosomal damage (clastogenic or aneugenic) in peripheral blood lymphocytes of operating-room nurses exposed to nitrous oxide. The study group comprised 46 women working at departments where the concentration of nitrous oxide ranged from 14 to 2308 mg/m3. The control population was composed of 28 women employed in the same hospitals but in non-surgical departments. The clastogenic/aneugenic effect of nitrous oxide was evaluated in lymphocytes using the standard micronucleus (MN) assay in combination with the fluorescence in situ hybridization (FISH) technique with pancentromeric probes. The results show a significant increase of the MN frequency in lymphocytes of exposed nurses compared with the control group (4.36+/-2.23 versus 9.02+/-4.67). The multiple regression analysis revealed a statistically significant relationship (p=0.0009) between MN frequency and exposure status, indicating that the level of exposure was the main factor affecting chromosomal damage. As assessed by FISH analysis, the overall frequencies of centromere-positive MN in the control and exposed groups were 43 and 49%, respectively. The increase observed in the exposed group may suggest a slight, statistically insignificant pro-aneugenic effect of exposure to nitrous oxide.