ABSTRACT
The use of nanostructured silica (SiO2) particles is no longer restricted to biomedical and (bio-) technological fields but rather finding applications in products of the food industry. Thus, our studies on the toxicological relevance of SiO2 nanoparticles focused on cytotoxic effects, the modulation of the cellular redox status and the impact on DNA integrity in human colon carcinoma cells (HT29). The results indicate that these SiO2 nanoparticles stimulate the proliferation of HT29 cells, depending on the incubation time and the particle size. The cytotoxicity of the investigated SiO2 nanoparticles was found to depend on the concentration, size and on the FCS content of the culture medium. Furthermore, SiO2 seem to interfere with glutathione biosynthesis. The results indicate further that effects of SiO2 NPs are not mediated by oxidative stress but by interference with the MAPK/ERK1/2 as well as the Nrf2/ARE signalling pathways. Additionally, investigations regarding DNA integrity revealed no substantial (oxidative) DNA damage.
Subject(s)
Colonic Neoplasms/drug therapy , Nanoparticles/administration & dosage , Silicon Dioxide/pharmacology , Blotting, Western , Cell Proliferation/drug effects , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Comet Assay , DNA Damage , Glutamate-Cysteine Ligase/metabolism , Glutathione/metabolism , HT29 Cells , Humans , L-Lactate Dehydrogenase/metabolism , MAP Kinase Signaling System/drug effects , Microscopy, Electron, Scanning , Mitochondria/drug effects , Mitochondria/metabolism , Oxidative Stress , Particle Size , Polymerase Chain Reaction , RNA/analysis , RNA/metabolism , Reactive Oxygen Species/metabolismABSTRACT
Single walled carbon nanotubes were studied with respect to cytotoxic and genotoxic properties in cells of the gastrointestinal tract as exemplified for the human colon carcinoma cell line HT29. No effect on cell growth in the sulphorhodamine B assay was observed after 24 h of incubation, whereas growth inhibitory properties were found after 48 and 72 h. After 24 h incubation a decrease of mitochondrial activity (WST-1) was measured (≥0.1 µg/ml), whereas membrane integrity (lactate dehydrogenase) was not affected. In cytotoxic concentrations, the formation of reactive oxygen species and a slight increase of total glutathione and nuclear Nrf2 were observed. However, already in subcytotoxic concentrations substantial DNA damaging effects were found in the alkaline comet assay, which were not associated with enhanced formation of formamidopyrimidine-DNA-glycosylase-sensitive sites. In addition, an increase of kinetochore-negative micronuclei (V79) and phosphorylation of the tumour suppressor protein p53 (HT29) underlined the genotoxic potential of these nanostructures.
Subject(s)
Colonic Neoplasms/pathology , DNA Damage , Nanotubes, Carbon/toxicity , Animals , Blotting, Western , Cell Line , Cell Proliferation , Colonic Neoplasms/enzymology , Colonic Neoplasms/metabolism , Comet Assay , Glutathione/metabolism , HT29 Cells , Humans , L-Lactate Dehydrogenase/metabolism , Mice , Micronucleus Tests , Microscopy, Atomic Force , Phosphorylation , Reactive Oxygen Species/metabolismABSTRACT
Three differently sized, highly dispersed platinum nanoparticle (Pt-NP) preparations were generated by supercritical fluid reactive deposition (SFRD) and deposited on a ß-cyclodextrin matrix. The average particle size and size distribution were steered by the precursor reduction conditions, resulting in particle preparations of <20, <100 and >100 nm as characterised by TEM and SEM. As reported previously, these Pt-NPs were found to cause DNA strand breaks in human colon carcinoma cells (HT29) in a concentration- and time-dependent manner and a distinct size dependency. Here, we addressed the question whether Pt-NPs might affect directly DNA integrity in these cells and thus behave analogous to platinum-based chemotherapeutics such as cisplatin. Therefore, DNA-associated Pt as well as the translocation of Pt-NPs through a Caco-2 monolayer was quantified by ICP-MS. STEM imaging demonstrated that Pt-NPs were taken up into HT29 cells in their particulate and aggregated form, but appear not to translocate into the nucleus or interact with mitochondria. The platinum content of the DNA of HT29 cells was found to increase in a time- and concentration-dependent manner with a maximal effect at 1,000 ng/cm(2). ICP-MS analysis of the cell culture medium indicated the formation of soluble Pt species, although to a limited extent. The observations suggest that DNA strand breaks mediated by metallic Pt-NPs are caused by Pt ions forming during the incubation of cells with these nanoparticles.
Subject(s)
DNA/metabolism , Enterocytes/drug effects , Intestinal Absorption , Metal Nanoparticles/toxicity , Mutagens/toxicity , Platinum/metabolism , Platinum/toxicity , Caco-2 Cells , Cell Polarity , Cell Proliferation/drug effects , Cell Survival/drug effects , Culture Media, Conditioned/chemistry , Cytoplasmic Vesicles/chemistry , Cytoplasmic Vesicles/ultrastructure , DNA/isolation & purification , Enterocytes/metabolism , Enterocytes/ultrastructure , HT29 Cells , Humans , Materials Testing , Metal Nanoparticles/chemistry , Metal Nanoparticles/ultrastructure , Mutagens/analysis , Mutagens/chemistry , Mutagens/metabolism , Organoplatinum Compounds , Osmolar Concentration , Particle Size , Platinum/analysis , Platinum/chemistry , Solubility , Time FactorsABSTRACT
Supercritical fluid reactive deposition was used for the deposition of highly dispersed platinum nanoparticles with controllable metal content and particle size distribution on beta-cyclodextrin. The average particle size and size distribution were steered by the precursor reduction conditions, resulting in particle preparations <20, <100, and >100 nm as characterized by transmission electron microscopy and scanning electron microscopy (SEM). These particle preparations of different size distributions were used to address the question as to whether metallic platinum particles are able to invade cells of the gastrointestinal tract as exemplified for the human colon carcinoma cell line HT29 and thus affect the cellular redox status and DNA integrity. Combined focused ion beam and SEM demonstrated that platinum nanoparticles were taken up into HT29 cells in their particulate form. The chemical composition of the particles within the cells was confirmed by energy-dispersive X-ray spectroscopy. The potential influence of platinum nanoparticles on cellular redoxsystems was determined in the DCF assay, on the translocation of Nrf-2 and by monitoring the intracellular glutathione (GSH) levels. The impact on DNA integrity was investigated by single cell gel electrophoresis (comet assay) including the formation of sites sensitive to formamidopyrimidine-DNA-glycosylase. Platinum nanoparticles were found to decrease the cellular GSH level and to impair DNA integrity with a maximal effect at 1 ng/cm(2). These effects were correlated with the particle size in an inverse manner and were enhanced with increasing incubation time but appeared not to be based on the formation of reactive oxygen species.
