Influence of serum on in situ proliferation and genotoxicity in A549 human lung cells exposed to nanomaterials

Abstract

In this work in situ proliferation of A549 human lung epithelial carcinoma cells exposed to nanomaterials (NMs) was investigated in the presence or absence of 10% serum. NMs were selected based on chemical composition, size, charge and shape (Lys-SiO2, TiO2, ZnO, and multi walled carbon nanotubes, MWCNTs). Cells were treated with NMs and 4h later, cytochalasin-B was added. 36h later, cell morphology was analyzed under a light microscope. Nuclearity was scored to determine the cytokinesis-block proliferation index (CBPI). CBPI, based on percentage of mono-, bi- and multi-nucleated cells, reflects cell toxicity and cell cycle delay. For some conditions depending on NM type (TiO2 and MWCNT) and serum concentration (0%) scoring of CBPI was impossible due to overload of agglomerated NMs. Moreover, where heavy agglomeration occurs, micronuclei (MN) detection and scoring under microscope was prevented. A statistically significant decrease of CBPI was found for ZnO NM suspended in medium in the absence or presence of 10% serum at 25μg/ml and 50μg/ml, respectively and for Lys-SiO2 NM at 3.5μg/ml in 0% serum. Increase in MN frequency was observed in cells treated in 10% serum with 50μg/ml ZnO. In 0% serum, the concentrations tested led to high toxicity. No genotoxic effects were induced by Lys-SiO2 both in the absence or presence of serum up to 5μg/ml. No toxicity was detected for TiO2 and MWCNTs in both 10% and 0% serum, up to the dose of 250μg/ml. Restoration of CBPI comparable to untreated control was shown for cells cultured without serum and treated with 5μg/ml of Lys-SiO2 NM pre-incubated in 100% serum. This observation confirms the protective effect of serum on Lys-SiO2 NM cell toxicity. In conclusion in situ CBPI is proposed as a simple preliminary assay to assess both NMs induced cell toxicity and feasibility of MN scoring under microscope.