Integrative approach in a safe by design context combining risk, life cycle and socio-economic assessment for safer and sustainable nanomaterials.

Moving towards safe and sustainable innovations is an international policy ambition. In the on-hand manuscript, a concept combining safe by design and sustainability was implemented through the integration of human and environmental risk assessment, life cycle assessment as well as an assessment of the economic viability. The result is a nested and iterative process in form of a decision tree that integrates these three elements in order to achieve sustainable, safe and competitive materials, products or services. This approach, embedded into the stage-gate-model for safe by design, allows to reduce the uncertainty related to the assessment of risks and impacts by improving the quality of the data collected along each stage. In the second part of the manuscript, the application is shown for a case study dealing with the application of nanoparticles for Li-Ion batteries. One of the general conclusions out of this case study is that data gaps are a key aspect in view of the reliability of the results.

Improving Quality in Nanoparticle-Induced Cytotoxicity Testing by a Tiered Inter-Laboratory Comparison Study.

The quality and relevance of nanosafety studies constitute major challenges to ensure their key role as a supporting tool in sustainable innovation, and subsequent competitive economic advantage. However, the number of apparently contradictory and inconclusive research results has increased in the past few years, indicating the need to introduce harmonized protocols and good practices in the nanosafety research community. Therefore, we aimed to evaluate if best-practice training and inter-laboratory comparison (ILC) of performance of the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay for the cytotoxicity assessment of nanomaterials among 15 European laboratories can improve quality in nanosafety testing. We used two well-described model nanoparticles, 40-nm carboxylated polystyrene (PS-COOH) and 50-nm amino-modified polystyrene (PS-NH2). We followed a tiered approach using well-developed standard operating procedures (SOPs) and sharing the same cells, serum and nanoparticles. We started with determination of the cell growth rate (tier 1), followed by a method transfer phase, in which all laboratories performed the first ILC on the MTS assay (tier 2). Based on the outcome of tier 2 and a survey of laboratory practices, specific training was organized, and the MTS assay SOP was refined. This led to largely improved intra- and inter-laboratory reproducibility in tier 3. In addition, we confirmed that PS-COOH and PS-NH2 are suitable negative and positive control nanoparticles, respectively, to evaluate impact of nanomaterials on cell viability using the MTS assay. Overall, we have demonstrated that the tiered process followed here, with the use of SOPs and representative control nanomaterials, is necessary and makes it possible to achieve good inter-laboratory reproducibility, and therefore high-quality nanotoxicological data.

Predicting the in vivo pulmonary toxicity induced by acute exposure to poorly soluble nanomaterials by using advanced in vitro methods.

BACKGROUND: Animal models remain at that time a reference tool to predict potential pulmonary adverse effects of nanomaterials in humans. However, in a context of reduction of the number of animals used in experimentation, there is a need for reliable alternatives. In vitro models using lung cells represent relevant alternatives to assess potential nanomaterial acute toxicity by inhalation, particularly since advanced in vitro methods and models have been developed. Nevertheless, the ability of in vitro experiments to replace animal experimentation for predicting potential acute pulmonary toxicity in human still needs to be carefully assessed. The aim of the study was to evaluate the differences existing between the in vivo and the in vitro approaches for the prediction of nanomaterial toxicity and to find advanced methods to enhance in vitro predictivity. For this purpose, rats or pneumocytes in co-culture with macrophages were exposed to the same poorly soluble and poorly toxic TiO2 and CeO2 nanomaterials, by the respiratory route in vivo or using more or less advanced methodologies in vitro. After 24 h of exposure, biological responses were assessed focusing on pro-inflammatory effects and quantitative comparisons were performed between the in vivo and in vitro methods, using compatible dose metrics. RESULTS: For each dose metric used (mass/alveolar surface or mass/macrophage), we observed that the most realistic in vitro exposure method, the air-liquid interface method, was the most predictive of in vivo effects regarding biological activation levels. We also noted less differences between in vivo and in vitro results when doses were normalized by the number of macrophages rather than by the alveolar surface. Lastly, although we observed similarities in the nanomaterial ranking using in vivo and in vitro approaches, the quality of the data-set was insufficient to provide clear ranking comparisons. CONCLUSIONS: We showed that advanced methods could be used to enhance in vitro experiments ability to predict potential acute pulmonary toxicity in vivo. Moreover, we showed that the timing of the dose delivery could be controlled to enhance the predictivity. Further studies should be necessary to assess if air-liquid interface provide more reliable ranking of nanomaterials than submerged methods.

Study of TiO2 P25 Nanoparticles Genotoxicity on Lung, Blood, and Liver Cells in Lung Overload and Non-Overload Conditions After Repeated Respiratory Exposure in Rats.

Inhaled titanium dioxide (TiO2) nanoparticles (NPs) can have negative health effects, and have been shown to cause respiratory tract cancer in rats. Inflammation has been linked to oxidative stress, and both have been described as possible mechanisms for genotoxicity of NPs, but rarely examined side-by-side in animal studies. In the present study, a wide range of complementary endpoints have been performed to study TiO2 P25 NP-induced genotoxicity in lung overload and non-overload conditions. Additionally, lung burden, inflammation, cytotoxicity and oxidative stress have also been evaluated in order to link genotoxicity with these responses. To assess quick and delayed responses after recovery, endpoints were evaluated at two time points: 2 h and 35 days after three repeated instillations. This study confirmed the previously described lung overload threshold at approximately 200-300 cm2 of lung burden for total particle surface area lung deposition or 4.2 µl/kg for volume-based cumulative lung exposure dose, above which lung clearance is impaired and inflammation is induced. Our results went on to show that these overload doses induced delayed genotoxicity in lung, associated with persistent inflammation only at the highest dose. The lowest tested doses had no toxicity or genotoxicity effects in the lung. In blood, no lymphocyte DNA damage, erythrocytes chromosomal damage or gene mutation could be detected. Our data also demonstrated that only overload doses induced liver DNA lesions irrespective of the recovery time. Tested doses of TiO2 P25 NPs did not induce glutathione changes in lung, blood or liver at both recovery times.

Air-liquid interface exposure to aerosols of poorly soluble nanomaterials induces different biological activation levels compared to exposure to suspensions.

BACKGROUND: Recently, much progress has been made to develop more physiologic in vitro models of the respiratory system and improve in vitro simulation of particle exposure through inhalation. Nevertheless, the field of nanotoxicology still suffers from a lack of relevant in vitro models and exposure methods to predict accurately the effects observed in vivo, especially after respiratory exposure. In this context, the aim of our study was to evaluate if exposing pulmonary cells at the air-liquid interface to aerosols of inhalable and poorly soluble nanomaterials generates different toxicity patterns and/or biological activation levels compared to classic submerged exposures to suspensions. Three nano-TiO2 and one nano-CeO2 were used. An exposure system was set up using VitroCell® devices to expose pulmonary cells at the air-liquid interface to aerosols. A549 alveolar cells in monocultures or in co-cultures with THP-1 macrophages were exposed to aerosols in inserts or to suspensions in inserts and in plates. Submerged exposures in inserts were performed, using similar culture conditions and exposure kinetics to the air-liquid interface, to provide accurate comparisons between the methods. Exposure in plates using classical culture and exposure conditions was performed to provide comparable results with classical submerged exposure studies. The biological activity of the cells (inflammation, cell viability, oxidative stress) was assessed at 24 h and comparisons of the nanomaterial toxicities between exposure methods were performed. RESULTS: Deposited doses of nanomaterials achieved using our aerosol exposure system were sufficient to observe adverse effects. Co-cultures were more sensitive than monocultures and biological responses were usually observed at lower doses at the air-liquid interface than in submerged conditions. Nevertheless, the general ranking of the nanomaterials according to their toxicity was similar across the different exposure methods used. CONCLUSIONS: We showed that exposure of cells at the air-liquid interface represents a valid and sensitive method to assess the toxicity of several poorly soluble nanomaterials. We underlined the importance of the cellular model used and offer the possibility to deal with low deposition doses by using more sensitive and physiologic cellular models. This brings perspectives towards the use of relevant in vitro methods of exposure to assess nanomaterial toxicity.

Oxidative stress pathways involved in cytotoxicity and genotoxicity of titanium dioxide (TiO2) nanoparticles on cells constitutive of alveolo-capillary barrier in vitro.

The health risks of nanoparticles remain a serious concern given their prevalence from industrial and domestic use. The primary route of titanium dioxide nanoparticle exposure is inhalation. The extent to which nanoparticles contribute to cellular toxicity is known to associate induction of oxidative stress. To investigate this problem further, the effect of titanium dioxide nanoparticles was examined on cell lines representative of alveolo-capillary barrier. The present study showed that all nanoparticle-exposed cell lines displayed ROS generation. Macrophage-like THP-1 and HPMEC-ST1.6R microvascular cells were sensitive to endogenous redox changes and underwent apoptosis, but not alveolar epithelial A549 cells. Genotoxic potential of titanium dioxide nanoparticles was investigated using the activation of γH2AX, activation of DNA repair proteins and cell cycle arrest. In the sensitive cell lines, DNA damage was persistent and activation of DNA repair pathways was observed. Moreover, western blot analysis showed that specific pathways associated with cellular stress response were activated concomitantly with DNA repair or apoptosis. Nanoparticles-induced oxidative stress is finally signal transducer for further physiological effects including genotoxicity and cytotoxicity. Within activated pathways, HSP27 and SAPK/JNK proteins appeared as potential biomarkers of intracellular stress and of sensitivity to endogenous redox changes, respectively, enabling to predict cell behavior.

Method development and inter-laboratory comparison about the determination of titanium from titanium dioxide nanoparticles in tissues by inductively coupled plasma mass spectrometry.

Nanosized titanium dioxide (TiO2) is one of the most interesting and valuable nanomaterials for the construction industry but also in health care applications, food, and consumer goods, e.g., cosmetics. Therefore, the properties associated with this material are described in detail. Despite its widespread use, the analytical determination and characterization of nanosized metal oxides is not as straightforward as the comparatively easy-to-detect metallic nanoparticles (e.g., silver or gold). This study presents the method development and the results of the determination of tissue titanium (Ti) levels after treatment of rats with the nanosized TiO2. Total Ti levels were chosen to evaluate the presence and distribution of TiO2 nanoparticles. A procedure consisting of incubation with a mixture of nitric acid (HNO3) and hydrofluoric acid (HF), and heating was developed to digest tissues and TiO2 nanomaterials in order to determine the total Ti content by inductively coupled plasma mass spectrometry (ICPMS). For the inter-laboratory comparison, altogether four laboratories analyzed the same samples upon digestion using the available ICPMS equipment. A major premise for any toxicokinetic study is the possibility to detect the chemical under investigation in biological samples (tissues). So, the study has to be performed with a dose high enough to allow for subsequent tissue level measurement of the chemical under investigation. On the other hand, dose of the chemical applied should not induce over toxicity in the animal as this may affect its absorption, distribution, metabolism, and excretion. To determine a non-toxic TiO2 dosage, an acute toxicity study in rats was performed, and the organs obtained were evaluated for the presence of Ti by ICPMS. Despite the differences in methodology and independent of the sample preparation and the ICPMS equipment used, the results obtained for samples with Ti concentrations >4 µg Ti/g tissue agreed well.

Pyrophosphorolysis-activated polymerization detects circulating tumor DNA in metastatic uveal melanoma.

PURPOSE: To develop a molecular tool to detect circulating tumor-derived DNA (ctDNA) in the plasma from patients with uveal melanoma as a marker of tumor burden and monitor treatment efficacy. EXPERIMENTAL DESIGN: A real-time PCR was developed on the basis of bidirectional pyrophosphorolysis-activated polymerization (bi-PAP) for the quantification of ctDNA using 3'blocked primer pairs specific for the 3 recurrent mutually exclusive mutations of Gα subunits GNAQ and GNA11. RESULTS: Sensitivity and specificity of bi-PAP were assessed on serial dilutions of tumor DNA in normal DNA for the 3 recurrent mutations. Each assay could detect a single mutated molecule per reaction, whereas 10(4) copies of normal DNA were not detected. The ctDNA was readily detected in plasma of mice bearing uveal melanoma xenografts in amounts proportional to circulating human DNA. Finally, plasma was almost always found positive (20 of 21 tested patients) in a prospective analysis of patients with metastatic uveal melanoma. CONCLUSIONS: Bi-PAP assays detect and quantify ctDNA in patients with metastatic uveal melanoma. A prospective study is ongoing to assess the clinical usefulness of ctDNA level in uveal melanoma.

Titanium dioxide nanoparticles induce DNA damage and genetic instability in vivo in mice.

Titanium dioxide (TiO(2)) nanoparticles are manufactured worldwide in large quantities for use in a wide range of applications including pigment and cosmetic manufacturing. Although TiO(2) is chemically inert, TiO(2) nanoparticles can cause negative health effects, such as respiratory tract cancer in rats. However, the mechanisms involved in TiO(2)-induced genotoxicity and carcinogenicity have not been clearly defined and are poorly studied in vivo. The present study investigates TiO(2) nanoparticles-induced genotoxicity, oxidative DNA damage, and inflammation in a mice model. We treated wild-type mice with TiO(2) nanoparticles in drinking water and determined the extent of DNA damage using the comet assay, the micronuclei assay, and the gamma-H2AX immunostaining assay and by measuring 8-hydroxy-2'-deoxyguanosine levels and, as a genetic instability endpoint, DNA deletions. We also determined mRNA levels of inflammatory cytokines in the peripheral blood. Our results show that TiO(2) nanoparticles induced 8-hydroxy-2'-deoxyguanosine, gamma-H2AX foci, micronuclei, and DNA deletions. The formation of gamma-H2AX foci, indicative of DNA double-strand breaks, was the most sensitive parameter. Inflammation was also present as characterized by a moderate inflammatory response. Together, these results describe the first comprehensive study of TiO(2) nanoparticles-induced genotoxicity in vivo in mice possibly caused by a secondary genotoxic mechanism associated with inflammation and/or oxidative stress. Given the growing use of TiO(2) nanoparticles, these findings raise concern about potential health hazards associated with TiO(2) nanoparticles exposure.

N-acetyl cysteine protects against ionizing radiation-induced DNA damage but not against cell killing in yeast and mammals.

Ionizing radiation (IR) induces DNA strand breaks leading to cell death or deleterious genome rearrangements. In the present study, we examined the role of N-acetyl-L-cysteine (NAC), a clinically proven safe agent, for it's ability to protect against gamma-ray-induced DNA strand breaks and/or DNA deletions in yeast and mammals. In the yeast Saccharomyces cerevisiae, DNA deletions were scored by reversion to histidine prototrophy. Human lymphoblastoid cells were examined for the frequency of gamma-H2AX foci formation, indicative of DNA double strand break formation. DNA strand breaks were also measured in mouse peripheral blood by the alkaline comet assay. In yeast, NAC reduced the frequency of IR-induced DNA deletions. However, NAC did not protect against cell death. NAC also reduced gamma-H2AX foci formation in human lymphoblastoid cells but had no protective effect in the colony survival assay. NAC administration via drinking water fully protected against DNA strand breaks in mice whole-body irradiated with 1Gy but not with 4Gy. NAC treatment in the absence of irradiation was not genotoxic. These data suggest that, given the safety and efficacy of NAC in humans, NAC may be useful in radiation therapy to prevent radiation-mediated genotoxicity, but does not interfere with efficient cancer cell killing.

Comparison of gene targeting efficiencies in two mosses suggests that it is a conserved feature of Bryophyte transformation.

The moss, Physcomitrella patens, is a novel tool in plant functional genomics due to its exceptionally high gene targeting efficiency that is so far unique for plants. To determine if this high gene targeting efficiency is exclusive to P. patens or if it is a common feature to mosses, we estimated gene-targeting efficiency in another moss, Ceratodon purpureus. We transformed both mosses with replacement vectors corresponding to the adenine phosphoribosyl transferase (APT) reporter gene. We achieved a gene targeting efficiency of 20.8% for P. patens and 1.05% for C. purpureus. Our findings support the hypothesis that efficient gene targeting could be a general mechanism of Bryophyte transformation.

MSH2 is essential for the preservation of genome integrity and prevents homeologous recombination in the moss Physcomitrella patens.

MSH2 is a central component of the mismatch repair pathway that targets mismatches arising during DNA replication, homologous recombination (HR) and in response to genotoxic stresses. Here, we describe the function of MSH2 in the moss Physcomitrella patens, as deciphered by the analysis of loss of function mutants. Ppmsh2 mutants display pleiotropic growth and developmental defects, which reflect genomic instability. Based on loss of function of the APT gene, we estimated this mutator phenotype to be at least 130 times higher in the mutants than in wild type. We also found that MSH2 is involved in some but not all the moss responses to genotoxic stresses we tested. Indeed, the Ppmsh2 mutants were more tolerant to cisplatin and show higher sensitivity to UV-B radiations. PpMSH2 gene involvement in HR was studied by assessing gene targeting (GT) efficiency with homologous and homeologous sequences. GT efficiency with homologous sequences was slightly decreased in the Ppmsh2 mutant compared with wild type. Strikingly GT efficiency with homeologous sequences decreased proportionally to sequence divergence in the wild type whereas it remained unaffected in the mutants. Those results demonstrate the role of PpMSH2 in the maintenance of genome integrity and in homologous and homeologous recombination.