Morphological observation of embryoid bodies completes the in vitro evaluation of nanomaterial embryotoxicity in the embryonic stem cell test (EST).

The wide and frequent use of engineered nanomaterials (NMs) raises serious concerns about their safety for human health. Our aim is to evaluate the embryotoxic potential of silver, uncoated and coated zinc oxide, titanium dioxide and silica NMs through the embryonic stem cell test (EST). EST is a validated in vitro assay that permits classification of chemicals into three classes (non, weakly or strongly embryotoxic). Because of the peculiar physico-chemical characteristics of NMs, we first adapted and simplified the differentiation protocol. To verify the efficiency of this adapted protocol we screened 3 well-characterized chemicals (5-fluorouracil, hydroxyurea and saccharin). Next, we assessed the embryotoxic potential of NMs. Our data showed that silver NM is classified as a strong embryotoxic compound, while coated and uncoated zinc oxide, titanium and silica NMs as weak embryotoxic compounds. In addition, we observed daily the formation and growth of embryoid bodies (EBs). We showed that multiple EBs formed in each well starting from 50 µg/ml of SiO2 while EB formation was inhibited starting from 20 µg/ml of ZnO NMs. This has never been reported with chemicals and could pose a risk of wrongly evaluating the NMs embryotoxic potential. For NMs, morphological observation of EBs can provide valuable information on early differentiation effects. Finally, we suggest that the prediction model should be revised for the assessment of NMs embryotoxicity.

Whole-mount in situ hybridization (WISH) optimized for gene expression analysis in mouse embryos and embryoid bodies.

Whole-mount in situ hybridization (WISH) is a technique widely used in developmental biology to study the localization of RNA sequences in intact tissues or whole organisms. In this chapter we present a detailed protocol that was optimized for gene expression analysis in early stage mouse embryos (5.5-10.5 days post-coitum) and embryoid bodies formed by differentiating embryonic stem cells and can be used for the detection of up to two distinct RNA sequences simultaneously. The initial steps of the procedure are the generation of the labeled riboprobe(s) and the embryo or embryoid body preparation, which can be completed in less than 2 days. The actual WISH procedure, comprised of the hybridization, the post-hybridization washes, and the immunological staining, can be completed in 3 days.

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

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-SiO(2), TiO(2), ZnO, and multi walled carbon nanotubes, MWCNTs). Cells were treated with NMs and 4h later, cytochalasin-B was added. 36 h 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 (TiO(2) 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 25 µg/ml and 50 µg/ml, respectively and for Lys-SiO(2) NM at 3.5 µg/ml in 0% serum. Increase in MN frequency was observed in cells treated in 10% serum with 50 µg/ml ZnO. In 0% serum, the concentrations tested led to high toxicity. No genotoxic effects were induced by Lys-SiO(2) both in the absence or presence of serum up to 5 µg/ml. No toxicity was detected for TiO(2) and MWCNTs in both 10% and 0% serum, up to the dose of 250 µg/ml. Restoration of CBPI comparable to untreated control was shown for cells cultured without serum and treated with 5 µg/ml of Lys-SiO(2) NM pre-incubated in 100% serum. This observation confirms the protective effect of serum on Lys-SiO(2) 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.

Methodological approaches influencing cellular uptake and cyto-(geno) toxic effects of nanoparticles.

Methods are needed to assess cytotoxicity and genotoxicity of nanoparticles (NPs). The influence of serum and the use of cytochalasin-B were assessed on the cellular uptake of amorphous silica NPs (SNPs) and their biological effects. Our observations indicate that some methodological approaches may modulate the outcome of the assay. Therefore the experimental design and choice of the assays are of great importance in nanotoxicology.