New methodological developments for testing the in vitro genotoxicity of nanomaterials: Comparison of 2D and 3D HepaRG liver cell models and classical and high throughput comet assay formats.

Nanomaterials (NMs) are defined as materials with at least one external dimension below 100 nm. Their small size confers them interesting unique physico-chemical properties, hence NMs are increasingly used in a diversity of applications. However, the specific properties of NMs could also make them more harmful than their bulk counterparts. Therefore, there is a crucial need to deliver efficient NM hazard assessment in order to sustain the responsible development of nanotechnology. This study analysed the genotoxic potential of several NMs: one titanium dioxide (TiO2) and two zinc oxide NMs (ZnO) that were tested up to 100 µg/mL on 2D and 3D hepatic HepaRG models. Genotoxicity analysis was performed comparing the alkaline comet assay in classical and high throughput formats. Moreover, oxidative DNA lesions were investigated with the Fpg-modified comet assay. Results showed that TiO2 NMs were not cytotoxic and not genotoxic in either cell model, although a small increase in the % tail DNA was observed in 3D HepaRG cells at 100 µg/mL in the classical format. The two ZnO NMs (ZnO S. NMs a commercial suspension and NM110 provided by the European Union Joint Research Centre) induced a concentration-dependent increase in cytotoxicity that was more pronounced in the 2D (>20% cytotoxicity was observed for ZnO S. at concentrations greater than 25 µg/mL, and for NM 110 at 50 µg/mL) than in the 3D model (more than 20% cytotoxicity for ZnO S. NMs at 50 µg/mL). While ZnO S. NMs induced DNA damage associated with cytotoxicity (at 25 and 50 µg/mL in 2D and 50 µg/mL in 3D), NM110 showed a clear genotoxic effect at non-cytotoxic concentrations (25 µg/mL in 2D and at 25 and 50 µg/mL in 3D). No major differences could be observed in the comet assay in the presence or absence of the Fpg enzyme. High throughput analysis using CometChip® mostly confirmed the results obtained with the classical format, and even enhanced the detection of genotoxicity in the 3D model. In conclusion, this study demonstrated that new approach methodologies (NAMs), 3D models and the high throughput format for the comet assay, were more efficient in the detection of genotoxic effects, and are therefore promising approaches to improve hazard assessment of NMs.

A comparison of control banding tools for nanomaterials.

Control banding (CB) is a useful approach to evaluate and control the risk of exposure to nanomaterials (NM) due to uncertainty surrounding their toxicity and challenges associated with their measurement. Four CB tools specifically developed for NMs (NanoSafer, Stoffenmanager-Nano, NanoTool, and the Precautionary matrix) have been evaluated for their changes to differences in hazard and exposure input data. The hazard and exposure classification were also compared with experimental data. The tools provided different hazard and emission/exposure outputs when compared with each other and with experimental data. For some of the tools the information required to estimate the hazard is not always available in the Safety Data Sheet and it requires expert judgement. The tools have the potential to be valuable starting points to assess areas of high priority, although outputs should be interpreted with care. Further work should be done to improve their estimates, especially the inclusion of modifiers that account for the effectiveness of the ventilation and the effect of high temperatures during the process.

Shape-Related Toxicity of Titanium Dioxide Nanofibres.

Titanium dioxide (TiO2) nanofibres are a novel fibrous nanomaterial with increasing applications in a variety of fields. While the biological effects of TiO2 nanoparticles have been extensively studied, the toxicological characterization of TiO2 nanofibres is far from being complete. In this study, we evaluated the toxicity of commercially available anatase TiO2 nanofibres using TiO2 nanoparticles (NP) and crocidolite asbestos as non-fibrous or fibrous benchmark materials. The evaluated endpoints were cell viability, haemolysis, macrophage activation, trans-epithelial electrical resistance (an indicator of the epithelial barrier competence), ROS production and oxidative stress as well as the morphology of exposed cells. The results showed that TiO2 nanofibres caused a cell-specific, dose-dependent decrease of cell viability, with larger effects on alveolar epithelial cells than on macrophages. The observed effects were comparable to those of crocidolite, while TiO2 NP did not decrease cell viability. TiO2 nanofibres were also found endowed with a marked haemolytic activity, at levels significantly higher than those observed with TiO2 nanoparticles or crocidolite. Moreover, TiO2 nanofibres and crocidolite, but not TiO2 nanoparticles, caused a significant decrease of the trans-epithelial electrical resistance of airway cell monolayers. SEM images demonstrated that the interaction with nanofibres and crocidolite caused cell shape perturbation with the longest fibres incompletely or not phagocytosed. The expression of several pro-inflammatory markers, such as NO production and the induction of Nos2 and Ptgs2, was significantly increased by TiO2 nanofibres, as well as by TiO2 nanoparticles and crocidolite. This study indicates that TiO2 nanofibres had significant toxic effects and, for most endpoints with the exception of pro-inflammatory changes, are more bio-active than TiO2 nanoparticles, showing the relevance of shape in determining the toxicity of nanomaterials. Given that several toxic effects of TiO2 nanofibres appear comparable to those observed with crocidolite, the possibility that they exert length dependent toxicity in vivo seems worthy of further investigation.

Quantum dot cytotoxicity in vitro: an investigation into the cytotoxic effects of a series of different surface chemistries and their core/shell materials.

The aim of this study was to assess the effects of a series of different surface coated quantum dots (QDs) (organic, carboxylated [COOH] and amino [NH2] polytethylene glycol [PEG]) on J774.A1 macrophage cell viability and to further determine which part of the QDs cause such toxicity. Cytotoxic examination (MTT assay and LDH release) showed organic QDs to induce significant cytotoxicity up to 48 h, even at a low particle concentration (20 nM), whilst both COOH and NH2 (PEG) QDs caused reduced cell viability and cell membrane permeability after 24 and 48 h exposure at 80 nM. Subsequent analysis of the elements that constitute the QD core, core/shell and (organic QD) surface coating showed that the surface coating drives QD toxicity. Elemental analysis (ICP-AES) after 48 h, however, also observed a release of Cd from organic QDs. In conclusion, both the specific surface coating and core material can have a significant impact on QD toxicity.

VEGF in the lung: a role for novel isoforms.

Vascular endothelial cell growth factor (VEGF) is a potent mitogen and permogen that increases in the plasma and decreases in the alveolar space in respiratory diseases such as acute respiratory distress syndrome (ARDS). This observation has led to controversy over the role of this potent molecule in lung physiology and disease. We hypothesized that some of the VEGF previously detected in normal lung may be of the anti-angiogenic family (VEGF(xxx)b) with significant potential effects on VEGF bioactivity. VEGF(xxx)b protein expression was assessed by indirect immunohistochemistry in normal and ARDS tissue. Expression of VEGF(xxx)b was also detected by immunoblotting in normal lung tissue, primary human alveolar type II (ATII) cells, and bronchoalveolar lavage (BAL) fluid in normal subjects and by ELISA in normal, "at risk," and ARDS subjects. The effect of VEGF(165) and VEGF(165)b on both human primary endothelial cells and alveolar epithelial cell proliferation was assessed by [(3)H]thymidine uptake. We found that VEGF(165)b was widely expressed in normal healthy lung tissue but is reduced in ARDS lung. VEGF(121)b and VEGF(165)b were present in whole lung, BAL, and ATII lysate. The proliferative effect of VEGF(165) on both human primary endothelial cells and human alveolar epithelial cells was significantly inhibited by VEGF(165)b (P < 0.01). These data demonstrate that the novel VEGF(xxx)b family members are expressed in normal lung and are reduced in ARDS. A specific functional effect on primary human endothelial and alveolar epithelial cells has also been shown. These data suggest that the VEGF(xxx)b family may have a role in repair after lung injury.

Failure to up-regulate VEGF165b in maternal plasma is a first trimester predictive marker for pre-eclampsia.

Pre-eclampsia is a pregnancy-related condition characterized by hypertension, proteinuria and endothelial dysfunction. VEGF(165)b, formed by alternative splicing of VEGF (vascular endothelial growth factor) pre-mRNA, inhibits VEGF(165)-mediated vasodilation and angiogenesis, but has not been quantified in pregnancy. ELISAs were used to measure means+/-S.E.M. plasma VEGF(165)b, sEng (soluble endoglin) and sFlt-1 (soluble fms-like tyrosine kinase-1). At 12 weeks of gestation, the plasma VEGF(165)b concentration was significantly up-regulated in plasma from women who maintained normal blood pressure throughout their pregnancy (normotensive group, 4.90+/-1.6 ng/ml; P<0.01, as determined using a Mann-Whitney U test) compared with non-pregnant women (0.40+/-0.22 ng/ml). In contrast, in patients who later developed pre-eclampsia, VEGF(165)b levels were lower than in the normotensive group (0.467+/-0.209 ng/ml), but were no greater than non-pregnant women. At term, plasma VEGF(165)b concentrations were greater than normal in both pre-eclamptic (3.75+/-2.24 ng/ml) and normotensive (10.58 ng/ml+/-3.74 ng/ml; P>0.1 compared with pre-eclampsia) pregnancies. Patients with a lower than median plasma VEGF(165)b at 12 weeks had elevated sFlt-1 and sEng pre-delivery. Concentrations of sFlt-1 (1.20+/-0.07 and 1.27+/-0.18 ng/ml) and sEng (4.4+/-0.18 and 4.1+/-0.5 ng/ml) were similar at 12 weeks of gestation in the normotensive and pre-eclamptic groups respectively. Plasma VEGF(165)b levels were elevated in pregnancy, but this increase is delayed in women that subsequently develop pre-eclampsia. In conclusion, low VEGF(165)b may therefore be a clinically useful first trimester plasma marker for increased risk of pre-eclampsia.

Dose-dependent effect of dehydroepiandrosterone, but not of its sulphate ester, on angiogenesis.

Although dehydroepiandrosterone (DHEA) is widely used in the elderly to prevent some adverse effects of ageing, possible deleterious side effects have not been fully assessed. We evaluated the direct actions of DHEA and DHEA sulphate on angiogenesis, a critical event in pathologies that are common in the elderly (cancer, atherosclerosis, inflammation... etc.). At physiological concentrations found in human plasma following DHEA therapy (1-50 nM), DHEA had no action on angiogenesis in vitro. In contrast, higher concentrations of DHEA (10-100 microM), which can be found in tissues after local administration or storage, inhibited in vitro endothelial cell proliferation (blockage in G2/M), migration and capillary tube formation and in vivo angiogenesis in the Matrigel plug assay. This inhibition might be due to a decreased glucose-6-phosphate dehydrogenase activity and to a modification of the tubulin network involved in cell proliferation and migration. The sulphate ester form of DHEA had no effect on angiogenesis.

Efficacy of dendrimer-mediated angiostatin and TIMP-2 gene delivery on inhibition of tumor growth and angiogenesis: in vitro and in vivo studies.

Gene transfer is an attractive approach to fight cancer by targeting cancer cells or their vasculature. Our study reports the inhibition of tumor growth and angiogenesis by a nonviral method using dendrimers associated with 36-mer anionic oligomers (ON36) for delivering angiostatin (Kringle 1-3) and tissue inhibitor of metalloproteinase (TIMP)-2 genes. The optimal concentrations of dendrimers and ON36 for an efficient green fluorescent protein (GFP) plasmid delivery in endothelial cells (HMEC-1) and cancer cells (MDA-MB-435) were first chosen. Then the efficacy of transfection was determined by testing angiostatin and TIMP-2 secretion by Western blot and the biologic effects were evaluated. Angiostatin gene transfer markedly reduced in vitro (i) HMEC-1 but not MDA-MB-435 proliferation; (ii) HMEC-1 and MDA-MB-435 wound healing reparation; and (iii) capillary tube formation. TIMP-2 gene transfer did not affect cell proliferation but strongly inhibited (i) wound healing of HMEC-1 and MDA-MB-435 cells; and (ii) capillary tube formation. Supernatants of transfected-MDA-MB-435 cells also inhibited the formation of angiogenic networks on Matrigel, indicating a paracrine effect. In vivo, intratumoral angiostatin or TIMP-2 gene delivery using dendrimers associated with ON36 effectively inhibited tumor growth by 71% and 84%, respectively. Combined gene transfer resulted in 96% inhibition of tumor growth. Tumor-associated vascularization was also greatly reduced. These findings provide a basis for the further development of nonviral delivery of genes to fight cancer.