This book addresses the gaps relating to health and safety issues of this field and aims to bring together fragmented knowledge on nanosafety. Not only do chapters address conventional toxicity issues, but also more recent developments such as food borne nanoparticles, life cycle analysis of nanoparticles and nano ethics. In particular this book presents a unique compilation of experimental and computational perspectives. The book is aimed towards postgraduates, academics, and practicing industry professionals but also serves as an excellent foundation for researchers new to nanotechnology and nanotoxicology.
About the Author
Professor Alok Dhawan is currently the Director at the Institute of Life Sciences in Ahmedabad University, India, on lien from CSIR-Indian Institute of Toxicology Research, Lucknow where he is Principal Scientist and Area Coordinator for the Nanomaterial Toxicology Group. Professor Dhawan is one of the originators of nanomaterial toxicology research in India. He is a Series Editor for the Royal Society of Chemistry Issues in Toxicology series.
Professor Rishi Shanker is also based with the Institute of Life Sciences at Ahmedabad University. Previous to this he was Chief Scientist at the CSIR-Indian Institute of Toxicology Research in Lucknow and a Professor at the Academy for Scientific and Innovative Research. Dr. Shanker served as Area Coordinator of core research areas of ‘Environmental Toxicology’ and ‘Nanomaterial Toxicology’ at CSIR-IITR.
Both Professor Dhawan and Professor Shanker were instrumental in the development of the state of art ‘Environmental Biotechnology’ facility at CSIR-NEERI.
Professor Diana Anderson is a Professor of Biomedical Science and Established Chair at the Bradford School of Medical Sciences, UK and a Distinguished Professor at Ahmedabad University. Professor Anderson is a Series Editor for the Current Toxicology series published by John Wiley and Sons, and the Editor in Chief of the Royal Society of Chemistry Issues in Toxicology series.
The Editors have published prolifically in the field of nanotoxicology individually and collaboratively. They are amongst the founding members of the Indian Nanoscience Society which launched in 2007 and have contributed realty to initiating this discipline in India.
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Nanotoxicology: Challenges for Biologists
ASHUTOSH KUMAR, SANJAY SINGH, RISHI SHANKER AND ALOK DHAWAN
The manufacture of nanoscale materials with novel physicochemical properties has led to powerful nanotechnology in the 21st century, which enables the potential of existing technologies to be realised. The uniqueness in the properties of these nanoscale materials continues to provide almost unlimited applications worldwide across engineering, medicine, agriculture, food industries and biotechnology. Today, there are more than 1800 nanoenabled consumer products are available in the public domain. The application of nanobased consumer products has also increased their inadvertent release into the environment during their production, usage, disposal and recycling. Living organisms including humans are exposed to these nanomaterials (NMs) throughout their life-cycle. Unfortunately, the information about human exposure and possible adverse health effects of NMs is still meagre. How properties of NMs define their interactions with cells, tissues and organs is a scientific challenge that must be addressed for the safe use of NMs.
Toxicity testing of NMs using existing in vitro and in vivo methods and models is a difficult task as there are so many different classes of NMs with various characteristics that can contribute to toxicity by diverse mechanisms. The characteristics such as NM size, shape, surface properties, composition, solubility, aggregation/agglomeration, particle uptake, the presence of mutagens and transition metals affiliated with the particles, etc. can influence the fate of NMs in biological systems. The most common underlying mechanisms of NM-induced toxicity are oxidative stress, inflammation, immunotoxicity and genotoxicity. NMs interact with the cells, tissues and organs of biological systems as they have a higher potential to move across the whole organism compared to bulk materials. Accumulation of NMs in their target organs can lead to cytotoxicity or genotoxicity. NMs can cross the blood-brain barrier, enter the blood or the central nervous system, with immense potential to directly affect cardiac and cerebral functions. The NMs also have the ability to redistribute in the biological system from their site of deposition and cause harmful effects. Therefore, it is prudent to understand the fate of NMs in biological systems. At present, the methods used for assessing the toxicity of chemicals in living systems, are used to evaluate the toxicity of NMs. However, several novel properties associated with the NMs make it imperative to develop new methods for measuring the toxicity of NMs. Therefore, in this chapter, an attempt has been made to address the different challenges in the toxicity assessment of NMs.
1.2 The Hurdles in Toxicity Evaluation of NMs
It is now well established that the properties of NMs are the combined function of their size, shape, surface area, surface-to-volume ratio, chemical composition, solubility and others. Hence, to study NMs' effects in living organisms and environments, the study design should be multipronged, and address NM characterization using validated protocols and hazard identification in humans and the environment. It is also important to mention that surface properties of NMs affect their biological behaviour. In order to measure the risk/toxicological endpoints associated with NMs, the material needs to be fully understood and characterized. Otherwise, the possible risk/ toxic effects cannot be easily attributed to a certain property of the NMs or even the NM itself. For example, impurities and other components could be responsible for the observed effects. Therefore, a critical assessment of the biological behaviour of NMs without a careful physicochemical characterization is not meaningful.
The physicochemical properties characterization of NMs includes a range of parameters such as the analysis of purity, crystallinity, solubility, chemical composition, surface chemistry, reactivity, size, shape, surface area, surface porosity, roughness and morphology. Changes in the elemental composition, size or surface properties of NMs can result in a transformation in physical and chemical properties:
Size: based on the material used in precursor solutions to produce NMs, properties such as solubility, transparency, absorption or emission wavelength, conductivity, melting point, colour and catalytic behaviour are changed by varying the particle size of NMs. Nanomaterials possess unique physicochemical properties due to their size; which also affects the mobility and transport behaviour of NMs.
Composition effects: it is clear that different particle compositions lead to different physical and chemical behaviours of the material.
Surface effects: the smaller the diameter of a spherical particle, the higher the surface-to-volume ratio and the specific surface area. This is accompanied by properties such as dispersity, conductivity, catalytic behaviour, chemical reactivity and optical properties. Therefore, more attention has to be paid to the surface material of a nanoparticle (NP) rather than its core material. When bare NMs come in contact with a heterogeneous environment, the smaller structures such as atoms, molecules or macromolecules attach to the surface of the NMs either by strong or weak interaction forces. In a biological environment, molecules such as proteins and polymers interact with the NM surface layer and form a "NM-protein corona". It has also been shown that it is not the NMs alone, but also the corona that defines the properties of the "particle-plus-corona" compound. This makes it necessary to understand not only the behaviour of NMs but also the biological interaction environment.
Agglomeration: agglomeration affects the surface properties of NMs and their bioavailability to the cells.
Solubility: some NMs are reported to produce ions in soluble form, which may be toxic to the cells e.g., ZnO, CuO.
Surface charge and dispersity: surface charge of the NMs affects the particle solubility in suspension, whereas the dispersity of NMs provides information about their tendency to agglomerate.
Dose metric: the exposure metric for NMs has been expressed, based on mass, number or surface area. The National Institute for Occupational Safety and Health (NIOSH) recommends that the "exposure metrics other than airborne mass concentration may be a better predictor of certain lung diseases, but it was decided that existing sampling methods will report in mass concentration because the toxicological effects observed are based on a mass dose". The issue of the proper metric for enumerating NPs in workplaces is still a debatable issue. As mentioned, surface area concentration has been found to correlate well, regardless of particle size, with pulmonary response. However, this may not be true for all particle types and may also be a function of the agglomeration state.
In brief, to assess the risk/toxicity of NMs, the primary criterion is to have full knowledge of the NMs to be tested. Considering the novel characteristics of NMs, unlike their chemical counterparts, it is imperative to undertake comprehensive characterization prior to risk/toxicity evaluation.
1.3 ENM Interference with Toxicity Test Methods
1.3.1 Interference of NPs with Metabolic Activity Detection Assays
The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay is a colorimetric test to determine the activity of cellular enzymes by the reduction of tetrazolium dye into its insoluble formazan crystals, which upon addition of dimethyl sulfoxide (DMSO) give a purple colour. The solubilized formazan absorbs at ~590 nm. Similarly, other related tetrazolium dyes such as 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5 -carboxanilide (XTT), 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl) -2-(4-sulfophenyl)-2H-tetrazolium (MTS) and the 8 (2-(2-methoxy-4-nitrophenyl)-3(4-nitrophenyl)-5-(2,4-disulfophenyl) -2H-tetrazolium (WSTs), are used in conjunction with the intermediate electron acceptor, 1-methoxy PMS. WST-1, a cell-impermeable dye. Reduction occurs outside the cell through the plasma membrane electron transport system and produces water-soluble formazan. These tests measure cellular metabolic activity via NAD(P)H-dependent cellular oxidoreductase enzymes, which under defined conditions, reflect the number of viable cells present in the test system. Toxicity tests of engineered nanomaterials (ENMs) have been frequently carried out by using these test systems. The interference of ENM dispersions with the optical detection of MTT-formazan have been observed with many ENM systems such as TiO2, ZnO NPs and carbon nanotubes (CNTs). ENMs having absorbance around the 500-600 nm (such as gold, silver and copper NPs) range are most likely to affect the absorbance by MTT-formazan in the ENPs-treated cells, whereas, MTT-formazan absorbance from untreated cells would not be affected, as they do not contain ENMs. Alternatively, ENMs having redox activity might undergo one-electron transition from many redox molecules (such as NAHP/NADPH, NAD/NADH and ADP/ATP), which ultimately may lead to the reduction of the MTT dye into MTT-formazan. Sometimes, it has been observed that certain lower concentrations of ENMs gives higher absorbance than corresponding controls. This may lead to the misinterpretation that exposure to ENMs can cause cell proliferation. However, the observed increase in absorbance is actually due to the reduction of more MTT-formazan dye by increased activity of mitochondrial dehydrogenase and other cellular oxidoreductase enzymes in the stressed cells on exposure to low concentrations of ENMs. Smaller ENMs (4-15 nm) composed of Au, Ag, AgO, Fe3O, CeO2 and CoO, have shown light absorption at the wavelengths used in most biological cytotoxicity test readouts: 340, 380, 405, 440, 540 and 550 nm. Thus, if these ENMs are toxic to cells, the decreased formazan formation (due to reduced cell metabolism) could be masked by the absorbance of these NMs due to their optical density, thus providing a false impression of lack of toxicity. Additionally, some ENMs can inhibit colour formation, thus exhibiting falsely a cytotoxic effect. In the case of CNTs it has been seen that CNTs absorb formazan molecules and protect them from being metabolized by cells. Under such circumstances, the decreased colour formation occurs due to the direct effect of CNTs on the MTT dye rather than a decrease in the number of living cells, thus leading to the false interpretation of a cytotoxic effect. Aluminium NPs also demonstrate a strong interaction with MTT dye resulting in significant misinterpretation of associated cytotoxicity.
Interference of NPs in Assays for Cell Death Measurement
Cell death measurement induced by the exposure of ENMs is usually measured by lactate dehydrogenase (LDH) quantification in cell supernatant. In principle, LDH reduces INT (2-(4-iodophenyl)-3-(4-nitrophenyl)5-phenyl tetrazolium chloride) in the presence of NADH + H+ (reduced β-nicotinamide adenine dinucleotide) to give a pink water-soluble formazan, which is quantified by light absorbance measurement in the visible region. The interference of ENM dispersions with the optical detection of INT might have happened due to the intrinsic absorbance of ENMs in the visible region (e.g., metallic NPs) and/or ENMs inducing reduction/oxidation under the influence of cellular biochemical reactions. Engineered nanoparticles (ENPs) may also react with INT leading to altered absorbance thus variability in assay outcomes. Some ENMs are highly catalytically active, thus may alter the intrinsic properties of assay reagents. Recently, experiments on copper-containing compounds, such as CuCl2, CuSO and Cu powder, showed interactions with LDH assay components. It was found that copper-containing compounds incubated with LDH showed inhibition of LDH calibrator detection depending on Cu salt dose. Recently, Kroll et al. reported that inhibition of the LDH assay in the presence of fine-sized ZnO NMs was dependent on the composition more than the size or surface. Han et al., found Ag NPs (~35 nm) deactivate LDH due to interaction of synthesis reagents with LDH whereas, TiO2 NPs (25 nm) were also found to interfere with the LDH assay due to adsorption of LDH molecules on the surface.
1.3.3 Interference of ENPs with Immunoassays
ENMs, due to their high surface area, are prone to adsorbing antibodies or other immunoassay components on their exposed surfaces. CNTs have been found to adsorb the antibodies on their surface, thus interfering with the assay results leading to misinterpretation. Similarly, Kroll et al. also reported TiO2 NPs as potential adsorbers of interleukins to their surfaces leading to a reduced level of IL-8 into dispersion. This was found to be concentration-dependent, where TiO2 concentrations below 10 µg cm-2 did not show any IL-8 adsorption. Other ENMs have also been reported to adsorb pro-inflammatory cytokines, for example metal oxides such as TiO2 and SiO2 are reported to adsorb IL-6, and carbon black (CB) to adsorb several different cytokines (GM-CSF, IL-8, IL-6, TNFα, TGFβ, etc.). The presence of serum in the experiment affects the result significantly. Kocbach et al. reported that cytokine binding was completely eliminated by adding serum proteins to the NP suspension. This may be due to the adsorption of serum proteins on NP eliminated, thus the formation of a protein corona and stabilization of NPs. Further, it was demonstrated by Brown et al. that an increase in NP concentration leads to the enhanced binding of cytokines on their surfaces.
1.3.4 Interference of ENMs in Assays with Enzymes
Several ENMs have been reported to interact with the enzymes of assay reagents. One such example reported by Kain et al. is the interaction of FPG (formamidopyrimidine-DNA glycosylase) that acts both as a N-glycosylase and an AP-lyase enzyme. Due to its N-glycosylase activity it releases damaged purines from double-stranded DNA, thus generating an apurinic (AP-site). Due to its AP-lyase activity it cleaves both 3' and 5' ends of the AP site thus leaving a one-base gap. Further, incorporation of FPG into the comet assay for DNA damage detection (see Section 3.10) has been shown to be a more accurate and reliable test for genotoxicity. Kain et al. conducted an experiment with a range of microparticles and NPs such as stainless steel, MnO2, Ag, CeO2, Co3O4, Fe3O4, NiO and SiO2 and followed the interactions of these particles and their released ions with FPG. Interestingly, they observed that incubation of these particles with FPG led to greatly decreased enzyme activity with Ag NPs, but also with CeO2, Co3O and SiO2 NPs. Further, studies have suggested that the decrease in enzymatic activity in the case of Ag NPs was mainly due to the Ag ions. However, in the case of CeO2, Co3O4 and SiO2 NPs, it was due to the physical adsorption of FPG on NP surfaces. Therefore, in the comet assay, the interaction of FPG with particles can lead to a decrease in the enzyme activity, thereby impairing the ability to detect genotoxicity. Further, this method may not be the most reliable method to assess the DNA damage potential of all ENMs. However, if used, other independent in vitro control methods should be used in parallel to measure genotoxicity.
1.3.5 Interference with Measurement of Free Radicals Generated due to ENM Exposure
The formation of free radicals under in vitro cell culture models due to ENM exposure is generally detected by a fluorescein derivative H2DCF-DA (2',7'-dicholorofluoresceindiacetate). The cell-permeable H2DCF-DA penetrates the cell membrane and is hydrolyzed by cellular esterases and converted via free radicals into the fluorescent oxidation product DCF. In a study by Kroll et al., when only ENMs (i.e., a cell-free system) were exposed to the substrate, H2DCF-DA, NPs were found to oxidize the substrate into fluorescent DCF. In this experiment, the interference of ENMs with the optical detection of DCF fluorescence was measured by replacing the assay substrate H2DCF-DA with defined amounts of fluorescent DCF in a cell-free system. They observed a reduction in DCF fluorescence transmission with all
24 types of ENMs tested from a particle concentration of 10 mg cm-2. The effect was most pronounced in the case of CB, which was explained on the basis of CB absorbance in the visible spectrum. Further, since excitation (~480 nm) and emission (~520 nm) of DCF lie within the spectrum of visible light, CB may absorb light from emitted DCF fluorescence and also from excitation energy, thereby interfering with the excitation wavelength of NPs. Other tested ENMs such as metal oxides, metal hydroxides and metal carbonates gave whitish or yellowish opaque dispersions in serum containing cell culture medium. Therefore, the decreased DCF fluorescence emission could be due to the large extent of light scattering, rather than absorption. The authors further reported that removing ENMs from suspension by washing or centrifugation before measuring the DCF fluorescence could be useful to avoid these methodological artefacts. In another experiment, Pfaller et al. reported enhanced fluorescence intensities when cell-free DCF assays were performed in the presence of 4.5 nm Au NPs. Such observations, could be due to the non-specific oxidation of H2DCF-DA into fluorescent DCF. This interference with DCF assays could lead to false-negative or false-positive results and an under-/over-estimation of ENM toxicity. Therefore, it is suggested that use of DCF assays for classical toxicology studies needs to be further optimized for each type of NP.
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Table of Contents
Nanotoxicology: Challenges for Biologists; Chemical Synthesis of Nanoparticles for Diverse Applications; Synthesis of Nanoparticles for Biomedical Applications; Protocols for In vitro and In vivo Toxicity Assessment of Engineered Nanoparticles; Nanoparticles in Biomedicine and Medicine, and Possible Clinical Toxicological Application of Peripheral Lymphocytes in the Risk Assessment Process for Susceptible Disease State Individuals; Health Hazard and Risk Assessment of Nanoparticles Applied in Biomedicine; Emerging Systems Toxicology Approaches in Nanosafety Assessment; Organ-on-chip Systems: An Emerging Platform for Toxicity Screening of Chemicals, Pharmaceuticals, and Nanomaterials; Progress Towards Risk Assessment for Engineered Nanomaterials; Three-dimensional Models for In vitro Nanotoxicity Testing; Computational Modelling of Biological Responses to Engineered Nanomaterials; Computational Approaches for Predicting Nanotoxicity at the Molecular Level; Safety Guidelines: Recommendations by Various Nations