Toxicity of carbon nanomaterials - Towards reliable viability assessment via new approach in flow cytometry

The scope of application of carbon nanomaterials in biomedical, environmental and industrial fields is recently substantially increasing. Since in vitro toxicity testing is the first essential step for any commercial usage, it is crucial to have a reliable method to analyze the potentially harmful effects of carbon nanomaterials. Even though researchers already reported the interference of carbon nanomaterials with common toxicity assays, there is still, unfortunately, a large number of studies that neglect this fact. In this study, we investigated interference of four bio-promising carbon nanomaterials (graphene acid (GA), cyanographene (GCN), graphitic carbon nitride (g-C3N4) and carbon dots (QCDs)) in commonly used LIVE/DEAD assay. When a standard procedure was applied, materials caused various types of interference. While positively charged g-C3N4 and QCDs induced false results through the creation of free agglomerates and intrinsic fluorescence properties, negatively charged GA and GCN led to false signals due to the complex quenching effect of the fluorescent dye of a LIVE/DEAD kit. Thus, we developed a new approach using a specific gating strategy based on additional controls that successfully overcame all types of interference and lead to reliable results in LIVE/DEAD assay. We suggest that the newly developed procedure should be a mandatory tool for all in vitro flow cytometry assays of any class of carbon nanomaterials.Web of Science2214art. no. 775

Carbon Nanodot:Supramolecular electron donor-acceptor hybrids featuring Perylenediimides

We describe the formation of charge-transfer complexes that feature electron-donating carbon nanodots (CND) and electron-accepting perylenediimides (PDI). The functionalities of PDIs have been selected to complement those of CNDs in terms of electrostatic and \u3c0-stacking interactions based on oppositely charged ionic head groups and extended \u3c0-systems, respectively. Importantly, the contributions from electrostatic interactions were confirmed in reference experiments, in which stronger interactions were found for PDIs that feature positively rather than negatively charged head groups. The electronic interactions between the components in the ground and excited state were characterized in complementary absorption and fluorescence titration assays that suggest charge-transfer interactions in both states with binding constants on the order of 8 7104\u2009M 121 (25\u2005L\u2009g 121). Selective excitation of the two components in ultrafast pump probe experiments gave a 210\u2005ps lived charge-separated state

Single exposure to aerosolized graphene oxide and graphene nanoplatelets did not initiate an acute biological response in a 3D human lung model

The increased mass production of graphene related materials (GRM), intended for a broad spectrum of applications, demands a thorough assessment of their potential hazard to humans and the environment. Particularly, the paramount concern has been expressed in regard to their interaction with the respiratory system in occupational exposure settings. It has been shown that GRM are easily respirable and can interact with lung cells resulting in the induction of oxidative stress or pulmonary inflammation. However, a comprehensive assessment of potential biological effects induced by GRM is currently hardly feasible to accomplish due to the lack of well-defined GRM materials and realistic exposure data. Herein, a 3D human lung model was combined with a commercial aerosolization system to study potential side effects of GRM. Two representative types of GRM were aerosolized onto the lung epithelial tissue surface. After 24 h post exposure, selected biological endpoints were evaluated, such as cell viability, morphology, barrier integrity, induction of (pro-)inflammation and oxidative stress reactions and compared with the reference material carbon black. Single exposure to all tested GRM at the two different exposure concentrations (∼300 and 1000 ng/cm2) did not initiate an observable adverse effect to the 3D lung model under acute exposure scenarios

Multiparameter toxicity assessment of novel DOPO-derived organophosphorus flame retardants

Halogen-free organophosphorus flame retardants are considered as replacements for the phased-out class of polybrominated diphenyl ethers (PBDEs). However, toxicological information on new flame retardants is still limited. Based on their excellent flame retardation potential, we have selected three novel 9,10- dihydro-9-oxa-10-phosphaphenanthrene-10-oxide (DOPO) derivatives and assessed their toxicological profile using a battery of in vitro test systems in order to provide toxicological information before their large-scale production and use. PBDE-99, applied as a reference compound, exhibited distinct neuroselective cytotoxicity at concentrations ≥10 μM. 6-(2-((6-Oxidodibenzo[1,2]oxaphosphinin-6-yl)amino)ethoxydibenzo[1,2]oxaphosphinine-6-oxide) (ETA-DOPO) and 6,6′-(ethane-1,2-diylbis(oxy))bis(dibenzo[1,2]oxaphosphinine-6-oxide) (EG-DOPO) displayed adverse effects at concentrations >10 μM in test systems reflecting the properties of human central and peripheral nervous system neurons, as well as in a set of non-neuronal cell types. DOPO and its derivative 6,6′-(ethane-1,2-diylbis(azanediyl))bis(6H-dibenzo[1,2]oxaphosphine-6-oxide) (EDA-DOPO) were neither neurotoxic, nor did they exhibit an influence on neural crest cell migration, or on the integrity of human skin equivalents.The two compounds furthermore displayed no inflammatory activation potential, nor did they affect algae growth or daphnia viability at concentrations ≤400 μM. Based on the superior flame retardation properties,biophysical features suited for use in polyurethane foams, and low cytotoxicity of EDA-DOPO, our results suggest that it is a candidate for the replacement of currently applied flame retardants

Interlaboratory comparison study of the Colony Forming Efficiency assay for assessing cytotoxicity of nanomaterials

Nanotechnology has gained importance in the past years as it provides opportunities for industrial growth and innovation. However, the increasing use of manufactured nanomaterials (NMs) in a number of commercial applications and consumer products raises also safety concerns and questions regarding potential unintended risks to humans and the environment. Since several years the European Commission’s Joint Research Centre (JRC) is putting effort in the development, optimisation and harmonisation of in vitro test methods suitable for screening and hazard assessment of NMs. Work is done in collaboration with international partners, in particular the Organisation for Economic Co-operation and Development (OECD). This report presents the results from an interlaboratory comparison study of the in vitro Colony Forming Efficiency (CFE) cytotoxicity assay performed in the frame of OECD's Working Party of Manufactured Nanomaterials (WPMN). Twelve laboratories from European Commission, France, Italy, Japan, Poland, Republic of Korea, South Africa and Switzerland participated in the study coordinated by JRC. The results show that the CFE assay is a suitable and robust in vitro method to assess cytotoxicity of NMs. The assay protocol is well defined and is easily and reliably transferable to other laboratories. The results obtained show good intra and interlaboratory reproducibility of the assay for both the positive control and the tested nanomaterials. In conclusion the CFE assay can be recommended as a building block of an in vitro testing battery for NMs toxicity assessment. It could be used as a first choice method to define dose-effect relationships for other in vitro assays.JRC.I.4-Nanobioscience

In vitro research reproducibility : Keeping up high standards

Concern regarding the reproducibility of observations in life science research has emerged in recent years, particularly in view of unfavorable experiences with preclinical in vivo research. The use of cell-based systems has increasingly replaced in vivo research and the application of in vitro models enjoys an ever-growing popularity. To avoid repeating past mistakes, high standards of reproducibility and reliability must be established and maintained in the field of in vitro biomedical research. Detailed guidance documenting the appropriate handling of cells has been authored but was received with quite disparate perception by different branches in biomedical research. In that regard, we intend to raise awareness of the reproducibility issue among scientists in all branches of contemporary life science research and their individual responsibility in this matter. We have herein compiled a selection of the most susceptible steps of everyday in vitro cell culture routines that have the potential to influence cell quality and recommend practices to minimize the likelihood of poor cell quality impairing reproducibility with modest investment of time and resources.publishe

Innovative Techniques and Strategies for a Reliable High-Throughput Genotoxicity Assessment

Damage to DNA is a central mechanism to the initiation of carcinogenesis. As a consequence, precise DNA damage detection is essential for an effective risk assessment of xenobiotics and constitutes a powerful tool for human biomonitoring and early stage cancer risk assessment. Here we highlight four innovative approaches for determining genotoxicity in a reliable and in the future high-throughput manner. In this context, we discuss and evaluate recent improvements to well-established methods and present promising new techniques

Assessing Genotoxicity of Ten Different Engineered Nanomaterials by the Novel Semi-Automated FADU Assay and the Alkaline Comet Assay

Increased engineered nanomaterial (ENM) production and incorporation in consumer and biomedical products has raised concerns about the potential adverse effects. The DNA damaging capacity is of particular importance since damaged genetic material can lead to carcinogenesis. Consequently, reliable and robust in vitro studies assessing ENM genotoxicity are of great value. We utilized two complementary assays based on different measurement principles: (1) comet assay and (2) FADU (fluorimetric detection of alkaline DNA unwinding) assay. Assessing cell viability ruled out false-positive results due to DNA fragmentation during cell death. Potential structure–activity relationships of 10 ENMs were investigated: three silica nanoparticles (SiO2-NP) with varying degrees of porosity, titanium dioxide (TiO2-NP), polystyrene (PS-NP), zinc oxide (ZnO-NP), gold (Au-NP), graphene oxide (GO) and two multi-walled carbon nanotubes (MWNT). SiO2-NPs, TiO2-NP and GO were neither cytotoxic nor genotoxic to Jurkat E6-I cells. Quantitative interference corrections derived from GO results can make the FADU assay a promising screening tool for a variety of ENMs. MWNT merely induced cytotoxicity, while dose- and time-dependent cytotoxicity of PS-NP was accompanied by DNA fragmentation. Hence, PS-NP served to benchmark threshold levels of cytotoxicity at which DNA fragmentation was expected. Considering all controls revealed the true genotoxicity for Au-NP and ZnO-NP at early time Points.publishe