RESUMEN
Crystalline silica has been classified as a human carcinogen, but there is still considerable controversy regarding its fibrogenic and carcinogenic potential. In the present study, we investigated the genotoxic potential of bentonite particles (diameter < 10 microm) with an a-quartz content of up to 6% and different chemical modifications (alkaline, acidic, organic). Human lung fibroblasts (IMR90) were incubated for 36 h, 48 h, or 72 h with bentonite particles in concentrations ranging from 1 to 15 microg/cm2. Genotoxicity was assessed using the micronucleus (MN) assay and kinetochore analysis. The generation of reactive oxygen species (ROS) caused by bentonite particles via Fenton-like mechanisms was measured acellularly using electron spin resonance (ESR) technique and intracellularly by applying an iron chelator. Our results show that bentonite-induced genotoxic effects in human lung fibroblasts are weak. The formation of micronuclei was only slightly increased after exposure of IMR90 cells to an acidic sample of bentonite dust with a quartz content of 4-5% for 36 h (15 microg/cm2), 48 h (5 microg/cm2), and 72 h (1 microg/cm2), to an alkaline sample with a quartz content of 5% for 48 h and 72 h (15 microg/cm2), and to an acidic bentonite sample with 1% quartz for 72 h (1 microg/cm2). Native (untreated) and organic activated bentonite particles did not show genotoxic effects in most of the experiments. Also, bentonite particles with a quartz content < 1% were negative in the micronucleus assay. Generation of ROS measured by ESR was dependent on the content of transition metals in the sample but not on the quartz content or the chemical modification. Reduction of MN after addition of the iron chelator 2,2'-dipyridyl showed that ROS formation also occurs intracellularly. Altogether, we conclude that the genotoxic potential of bentonite particles is generally low but can be altered by the content of quartz and available transition metals.
Asunto(s)
Bentonita/toxicidad , Cinetocoros , Pulmón/efectos de los fármacos , Pruebas de Micronúcleos , Cuarzo/análisis , Bentonita/análisis , Células Cultivadas , Espectroscopía de Resonancia por Spin del Electrón , Fibroblastos/efectos de los fármacos , Humanos , Radical Hidroxilo , Quelantes del Hierro/farmacología , Cinetocoros/efectos de los fármacos , Especies Reactivas de OxígenoRESUMEN
Considering the biological reactivity of pure quartz in lung cells, there is a strong interest to clarify the cellular effects of respirable siliceous dusts, like bentonites. In the present study, we investigated the cellular uptake and the cytotoxic potential of bentonite particles (Ø< 10 microm) with an alpha-quartz content of up to 6% and different chemical modifications (activation: alkaline, acidic, organic) in human lung fibroblasts (IMR90). Additionally, the ability of the particles to induce apoptosis in IMR90-cells and the hemolytic activity was tested. All bentonite samples were tested for endotoxins with the in vitro-Pyrogen test and were found to be negative. Cellular uptake of particles by IMR90-cells was studied by transmission electron microscopy (TEM). Cytotoxicity was analyzed in IMR90-cells by determination of viable cells using flow cytometry and by measuring of the cell respiratory activity. Induced apoptotic cells were detected by AnnexinV/Propidiumiodide-staining and gel electrophoresis. Our results demonstrate that activated bentonite particles are better taken up by IMR90-cells than untreated (native) bentonite particles. Also, activated bentonite particles with a quartz content of 5-6% were more cytotoxic than untreated bentonites or bentonites with a quartz content lower than 4%. The bentonite samples induced necrotic as well as apoptotic cell death. In general, bentonites showed a high membrane-damaging potential shown as hemolytic activity in human erythrocytes. We conclude that cellular effects of bentonite particles in human lung cells are enhanced after chemical treatment of the particles. The cytotoxic potential of the different bentonites is primarily characterized by a strong lysis of the cell membrane.