Effects on male reproductive cells (TM3 Leydig and TM4 Sertoli).

<p>The cell viability by WST-1 assay was evaluated in TM3 cells (A, B and C) and TM4 cells (D, E and F) following the exposure for 24h to TiO_<b>2</b> NM-103 and NM-104, ZnO NM-110 and NM-111 and SiO2 NM-200 and NM-203. The ranges of doses used were between 0.125 μg/ml (0.037 μg/cm²) and 200 μg/ml (60.24 μg/cm²). WST-1 assay measurements were taken (120 min post addition). All results were corrected for WST-1, as the control showed presence of interference in absorbance reading as a result of WST. The statistical analysis of the data (n = 6) was performed by one way ANOVA, Dunnett's multiple comparison test and Tukey’s post-hoc test (p<0.001*).</p

Effects on immune cells (MH-S murine alveolar macrophages).

<p>Comparative evaluation of cell viability evaluated by resazurin assay (A and B) and NRU assay (C and D) after exposure to ZnO (NM-110 and NM-111). Cells were grown for 24h in complete growth medium and then exposed for 24h, 48h and 72h to different concentrations of ZnO NMs. At the end of the incubation, cell viability was assessed using two different assays (resazurin and NRU). Data are means ± SD of 10 independent determinations in two separate experiments. Statistical analysis was performed using one-way ANOVA followed by Bonferroni post-hoc test. *p< 0.05, **p< 0.01, ***p< 0.001.</p

Effects on the airway epithelial cell barrier formed by Calu-3 cells.

<p>Evaluation of TEER during the exposure to TiO2 NM-103 (A) and NM-104 (B), ZnO NM-110 (C) and NM-111 (D) and SiO2 NM-200 (E) and NM-203 (F). Calu-3 epithelial cells were grown for 10 days in a double-chamber culture system to form a tight monolayer. The epithelial monolayer was then exposed at the apical side to different concentrations of NMs: 128 μg/ml (80 μg/cm²) of TiO2 and SiO2, 32 μg/ml (20 μg/cm²) and 64 μg/ml (40 μg/cm²) of ZnO. The trans-epithelial electrical resistance (TEER) was measured at the indicated times with a voltohmeter. Data are values obtained from 12 monolayers used in three separate experiments and are expressed as % of the initial value ± SD (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127174#sec019" target="_blank">Methods</a>). Statistical analysis was performed using two-way ANOVA followed by Bonferroni post-hoc test. **p< 0.01, ***p< 0.001.</p

Methodology applied for the in vitro toxicity assessment.

<p>Ten different assays were used for the evaluation of cytotoxicity (resazurin, NRU, WST-1, WST-8, LDH and CFE), embryotoxicity (EST), epithelial integrity (TEER), cytokine secretion (ELISA) and oxidative stress (DCF) on twelve different cellular systems.</p

Effects on immune cells (primary human monocyte-derived macrophages).

<p>Cell viability was evaluated using LDH assay after 24h of exposure to TiO_<b>2</b> NM-103 and NM-104 (A), ZnO NM-110 and NM-111 (B) or SiO_<b>2</b> NM-200 and NM-203 (C). The results are expressed as % of cell viability (mean ± SD) versus control cells (100%) and are obtained from three to four independent experiments (donors). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test (*p< 0.05, **p < 0.01, ***p < 0.001).</p

Effects on the gastrointestinal tract (Caco-2 intestinal cells).

<p>The cytotoxic and cytostatic effects were evaluated by Colony Forming Efficiency Assay (CFE) in Caco-2 cells exposed to 100 μg/ml of TiO_<b>2</b> (NM-103 and NM-104) (A) and SiO_<b>2</b> (NM-200 and NM-203) (B) for 3 (short exposure) and 10 days (long term, repeated-dose exposure). The results are expressed as CFE (% of solvent control) = ([average No. of treatment colonies/average No. of solvent control colonies] × 100). The solvent control (0.05% BSA) did not induce cytotoxicity. Data are reported as means ± SEM (standard error mean = standard deviation/√number of replicates). One-way analysis of variance (ANOVA) with post hoc test (Dunnett's Multiple Comparison Test) for comparing groups of data versus one control group was used (** p<0.01; *** p<0.001).</p

Effects on immune cells (RAW 264.7 murine macrophages).

<p>Comparative evaluation of cell viability by resazurin assay (A and B) and NRU assay (C and D) after exposure to ZnO (NM-110 and NM-111). Cells were grown for 24h in complete growth medium and then exposed for 24h, 48h and 72h to different concentrations of ZnO NMs. At the end of the incubation, cell viability was assessed using two different assays (resazurin and NRU). Data are means ± SD of 10 independent determinations in two separate experiments. Statistical analysis was performed using one-way ANOVA followed by Bonferroni post-hoc test. *p< 0.05, **p< 0.01, ***p< 0.001.</p