Mary Gulumian

The limits of testing particle-mediated oxidative stress in vitro in predicting diverse pathologies; relevance for testing of nanoparticles.

In vitro studies with particles are a major staple of particle toxicology, generally used to investigate mechanisms and better understand the molecular events underlying cellular effects. However, there is ethical and financial pressure in nanotoxicology, the new sub-specialty of particle toxicology, to avoid using animals. Therefore an increasing amount of studies are being published using in vitro approaches and such studies require careful interpretation. We point out here that 3 different conventional pathogenic particle types, PM10, asbestos and quartz, which cause diverse pathological effects, have been reported to cause very similar oxidative stress effects in cells in culture. We discuss the likely explanation and implications of this apparent paradox, and its relevance for testing in nanotoxicology.

Mechanistically Identified Suitable Biomarkers of Exposure, Effect, and Susceptibility for Silicosis and Coal-Worker'S Pneumoconiosis: A Comprehensive Review

Clinical detection of silicosis is currently dependent on radiological and lung function abnormalities, both late manifestations of disease. Markers of prediction and early detection of pneumoconiosis are imperative for the implementation of timely intervention strategies. Understanding the underlying mechanisms of the etiology of coal workers pneumoconiosis (CWP) and silicosis was essential in proposing numerous biomarkers that have been evaluated to assess effects following exposure to crystalline silica and/or coal mine dust. Human validation studies have substantiated some of these proposed biomarkers and argued in favor of their use as biomarkers for crystalline silica- and CWP-induced pneumoconiosis. A number of “ideal” biological markers of effect were identified, namely, Clara cell protein-16 (CC16) (serum), tumor necrosis factor-α (TNF-α) (monocyte release), interleukin-8 (IL-8) (monocyte release), reactive oxygen species (ROS) measurement by chemiluminescence (neutrophil release), 8-isoprostanes (serum), total antioxidant levels measured by total equivalent antioxidant capacity (TEAC), glutathione, glutathione peroxidase activity, glutathione S-transferase activity, and platelet-derived growth factor (PDGF) (serum). TNF-α polymorphism (blood cellular DNA) was identified as a biomarker of susceptibility. Further studies are planned to test the validity and feasibility of these biomarkers to detect either high exposure to crystalline silica and early silicosis or susceptibility to silicosis in gold miners in South Africa.

Dissolution and biodurability: Important parameters needed for risk assessment of nanomaterials.

Biopersistence and biodurability have the potential to influence the long-term toxicity and hence pathogenicity of particles that deposit in the body. Therefore, biopersistence and biodurability are considered to be important parameters needed for the risk assessment of particles and fibres. Dissolution, as a measure of biodurability, is dependent on the chemical and physical properties (size, surface area, etc.) of particles and fibres and also of the suspension medium including its ionic strength, pH, and temperature. In vitro dissolution tests can provide useful insights as to how particles and fibres may react in biological environments; particles and fibres that release ions at a higher rate when suspended in vitro in a specific simulated biological fluid will be expected to do so when they exist in a similar biological environment in vivo. Dissolution of particles and fibres can follow different reaction kinetics. For example, the majority of micro-sized particles and fibres follow zero-order reaction kinetics. In this case, although it is possible to calculate the half-time of a particle or fibre, such calculation will be dependent on the initial concentration of the investigated particle or fibre. Such dependence was eliminated in the shrinking sphere and fibre models where it was possible to estimate the lifetimes of particles and fibres as a measure of their biodurability. The latter models can be adapted for the dissolution studies of nanomaterials. However, the models may apply only to nanomaterials where their dissolution follows zero-order kinetics. The dissolution of most nanomaterials follows first-order kinetics where dependence on their initial concentration of the investigated nanomaterials is not required and therefore it is possible to estimate their half-times as a measure of their biodurability. In dissolution kinetics for micro-sized and nano-sized particles and fibres, knowledge of dissolution rate constants is necessary to understand biodurability. Unfortunately, many studies on dissolution of nanoparticles and nanofibres do not determine the dissolution rates and dissolution rate constants. The recommendation is that these parameters should be considered as part of the important descriptors of particle and fibre physicochemical properties, which in turn, will enable the determination of their biodurability.

Challenges facing sterilization and depyrogenation of nanoparticles: Effects on structural stability and biomedical applications

This review outlines and compares techniques that are currently available for the sterilization of nanoparticles and addresses the topic of endotoxin contamination. Several techniques are available for the removal of microbial contamination from nanoparticles developed for use in nanomedicine applications. These techniques include filtration, autoclaving and irradiation, as well as formaldehyde, ethylene oxide and gas plasma treatments. Of these sterilization methodologies, filtration may potentially remove microbial contamination without altering the physicochemical properties of the carrier nanoparticles, nor affecting their toxicity and functionality. However, no single process may be applied to all nanoparticle preparations and, therefore, it is recommended that each nanoparticle-drug system be validated on a case-by-case basis. From the clinical editor: This comprehensive review covers the currently available methods for removal of microbial contaminations from nanoparticles for nanomedicine applications. The review highlights the pros and cons of each available method. Authors conclude that there is no single best method and recommend a customized approach for each nanoparticle system.

Label-free in vitro toxicity and uptake assessment of citrate stabilised gold nanoparticles in three cell lines.

BackgroundReliable in vitro toxicity testing is needed prior to the commencement of in vivo testing necessary for hazard identification and risk assessment of nanoparticles. In this study, the cytotoxicity and uptake of 14 nm and 20 nm citrate stabilised gold nanoparticles (AuNPs) in the bronchial epithelial cell line BEAS-2B, the Chinese hamster ovary cell line CHO, and the human embryonic kidney cell line HEK 293 were investigated.MethodsCytotoxicity of the AuNPs was assessed via traditional XTT-, LDH-, and ATP-based assays, followed by cell impedance studies. Dark-field imaging and hyperspectral imaging were used to confirm the uptake of AuNPs into the cells.ResultsInterference of the AuNPs with the XTT- and ATP-based assays was overcome through the use of cell impedance technology. AuNPs were shown to be relatively non-toxic using this methodology; nevertheless CHO cells were the most sensitive cell type with 20 nm AuNPs having the highest toxicity. Uptake of both 14 nm and 20 nm AuNPs was observed in all cell lines in a time- and cell type-dependent manner.ConclusionsUsing the cell impedance and dark-field hyperspectral imaging technologies, it was possible to study the toxicity of AuNPs in different cell lines and show that these cells could internalize AuNPs with their subsequent intracellular aggregation. It was also possible to show that this toxicity would not correlate with the level of uptake but it would correlate with cell-type and the size of the AuNPs. Therefore, these two label-free methodologies used in this study are suitable for in vitro studies on the effects of AuNPs, and could present themselves as appropriate and valuable methodologies for future nanoparticle toxicity and uptake studies.

Developmental toxicity of engineered nanoparticles

Publisher Summary This chapter discusses developmental toxicity of engineered nanoparticles. Revolutionary developments of physics, chemistry and material sciences have led to the emergence of nanotechnology. Nano-objects have found different applications as diagnostic and therapeutic tools in biomedicine. Engineered nanoparticles are defined by differences in their shape, size, surface charge and chemical composition, mostly due to the mode of their production. Some of the nanoparticles are carbon nanoparticle, carbon nanotubes, quantum dots, inorganic nanoparticles and inorganic nanotubes. Non-degradable nanomaterials can accumulate in organs and inside cells where they can exert detrimental effects. One major uncertainty in interpretation of experimental toxicity studies as well as in risk assessment of engineered nanomaterial arises from lack of systematic knowledge about the physico-chemical characteristics of the material arriving at the major portals through which nanoparticles can enter the body, i.e. lung, skin, gastro-intestinal tract, nasal olfactory structures and eyes. The small size of nanoparticles allows them to easily enter and traverse tissues, cells and organelles since the actual size of engineered nanoparticles is similar to that of many biological molecules and structures. Nanoparticles are translocated from lung to blood and across the blood–brain barrier as well as the placenta. Maternal exposure to engineered nanoparticles may potentially affect fetal development directly as well as through indirect pathways. Engineered nanomaterials can cause inflammation, allergy, genotoxicity and carcinogenicity. The need for risk assessment of engineered nanomaterials has also generated a need for a novel risk assessment concept. The goal of control banding is to prevent excessive exposure to compounds such as engineered nanomaterials.

An Update on the Detoxification Processes for Silica Particles and Asbestos Fibers: Successess and Limitations

Inhalation of asbestos fibers and crystalline silica produces a number of diseases including fibrosis and cancer. Investigations into the mechanisms involved in mineral particle-induced toxicity indicated the importance of their surfaces in the pathological consequences. Masking of the surface sites has therefore featured prominently in a number of detoxification processes that have been investigated. The majority of the detoxification processes were, however, conducted to elucidate the involvement of a particular surface site in the toxicity of a specific mineral. Others were investigated with the aim of large industrial applications to be applied during mining, handling, processing, transporting, and disposing of minerals. It can be concluded that, to date, there is no single detoxification process that could be applied universally to all different types of mineral particles. Those that have shown some success could not completely abolish all adverse effects. Further elucidation of mechanisms of particle-induced toxicity may open new possibilities for detoxification processes. The author thanks Professor J. C. A. Davies for his constructive comments and for proofreading the manuscript of this article.

Evaluation of the phenolic and flavonoid contents and radical scavenging activity of three southern African medicinal plants

Warburgia salutaris (Bertol. F.) Chiovs, Rhoicissus tridentata (L.f.) Wild & Drum and Terminalia sericea (Burch. ex DC.), are widely used medicinal plants in southern Africa. The aim of the study was to determine the phenolic and flavonoid content and evaluate the antioxidant activity of the three medicinal plants. Total phenolic and flavonoid contents were determined spectrophotometrically as gallic acid and rutin equivalents, respectively. Individual phenolic acids were identified by means of gas chromatography-mass spectrometry. Antioxidant activities of the crude extracts were assessed using the TEAC assay. The highest phenolic content was detected in the crude methanol extract of the bark of W. salutaris and the highest flavonoid content was found in the crude methanol extract of the leaves of this plant. In all the studied plants the alkaline hydrolysable fraction yielded a greater variety of phenolic acids compared to the soluble/free phenolic acid fractions. The three medicinal plants investigated were found to be strong radical scavengers supporting the traditional use of these medicinal plants.

From the Cover: An Investigation of the Genotoxicity and Interference of Gold Nanoparticles in Commonly Used In Vitro Mutagenicity and Genotoxicity Assays

The suitability of 4 in vitro assays, commonly used for mutagenicity and genotoxicity assessment, was investigated in relation to treatment with 14 nm citrate-stabilized gold nanoparticles (AuNPs). Specifically, the Ames test was conducted without metabolic activation, where no mutagenic effects were observed. High resolution transmission electron microscopy and Cytoviva dark-field image analysis showed that AuNPs did not enter the bacterial cells, thus confirming the unreliability of the Ames test for nanoparticle mutagenicity studies. In addition, the Chinese hamster ovary (CHO) cell line was used for Comet, Chromosome aberration and Micronucleus assays. CHO cells were treated with AuNPs for 20 h at 37 °C. Cytotoxicity was not detected by cell impedance studies even though AuNP uptake was confirmed using Cytoviva image analysis. The DNA damage was statistically significant in treated cells when assessed by the Comet assay. However, minimal and nonstatistically significant chromosomal DNA damage was observed using the chromosome aberration and micronucleus assays. In this study, we showed that false positive results obtained with Comet assay may have been due to the possibility of direct contact between the residual, intracellular AuNPs and DNA during the assay procedure. Therefore, the chromosome aberration and micronucleus assays are better suited to assess the genotoxic effects of nanoparticles due to low probability of such direct contact occurring. Genotoxic effect of 14 and 20 nm citrate-stabilized, as well as, 14 nm PCOOH AuNPs were also investigated using chromosome aberration and micronucleus assays. Based on our acceptance criteria for a positive genotoxic response, none of the AuNPs were found to be genotoxic in either of these assays.

Approaches to Develop Alternative Testing Strategies to Inform Human Health Risk Assessment of Nanomaterials

The development of alternative testing strategies (ATS) for hazard assessment of new and emerging materials is high on the agenda of scientists, funders, and regulators. The relatively large number of nanomaterials on the market and under development means that an increasing emphasis will be placed on the use of reliable, predictive ATS when assessing their safety. We have provided recommendations as to how ATS development for assessment of nanomaterial hazard may be accelerated. Predefined search terms were used to identify the quantity and distribution of peer-reviewed publications for nanomaterial hazard assessment following inhalation, ingestion, or dermal absorption. A summary of knowledge gaps relating to nanomaterial hazard is provided to identify future research priorities and areas in which a rich data set might exist to allow ATS identification. Consultation with stakeholders (e.g., academia, industry, regulators) was critical to ensure that current expert opinion was reflected. The gap analysis revealed an abundance of studies that assessed the local and systemic impacts of inhaled particles, and so ATS are available for immediate use. Development of ATS for assessment of the dermal toxicity of chemicals is already relatively advanced, and these models should be applied to nanomaterials as relatively few studies have assessed the dermal toxicity of nanomaterials to date. Limited studies have investigated the local and systemic impacts of ingested nanomaterials. If the recommendations for research prioritization proposed are adopted, it is envisioned that a comprehensive battery of ATS can be developed to support the risk assessment process for nanomaterials. Some alternative models are available for immediate implementation, while others require more developmental work to become widely adopted. Case studies are included that can be used to inform the selection of alternative models and end points when assessing the pathogenicity of fibers and mode of action of nanomaterial toxicity.