Chapter 1: Nanotoxicology: Challenges for Biologists
Published:03 Nov 2017
A. Kumar, S. Singh, R. Shanker, and A. Dhawan, in Nanotoxicology: Experimental and Computational Perspectives, ed. A. Dhawan, D. Anderson, and R. Shanker, The Royal Society of Chemistry, 2017, ch. 1, pp. 1-16.
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Nanotechnology is a rapidly developing field due to the amalgamation of different science disciplines such as physical, chemical, biological and engineering. The unique size-dependent properties of nanomaterials make them superior and indispensable in many areas of human activity. Today, more than 1800 nano-enabled consumer products are available across engineering, medicine, agriculture, food industries and biotechnology sectors. However, nanoparticles are being incorporated into commercial products at a faster rate than the development of knowledge and regulations to mitigate potential health and environmental impacts associated with their manufacture, application and disposal. At present, the methods used for assessing the toxicity of chemicals in living systems, are used to evaluate the toxicity of nanoparticles. However, several novel properties associated with nanoparticles are responsible for false results. Also the reproducibility of the data in nanotoxicity-based experiments is a big concern. Therefore, in this chapter, an attempt is made to address the different challenges in the toxicity assessment of nanoparticles.
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 nano-enabled consumer products are available in the public domain.1 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.2,3 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.4
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.5–8 can influence the fate of NMs in biological systems.9 The most common underlying mechanisms of NM-induced toxicity are oxidative stress, inflammation, immunotoxicity and genotoxicity.10 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.11 Accumulation of NMs in their target organs can lead to cytotoxicity or genotoxicity.12 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.13 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.14 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.15,16 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”.17 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).18 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 activity19 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, Fe3O4, 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.20 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.20 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.21 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.22
1.3.2 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.23 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.24 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, CuSO4 and Cu powder, showed interactions with LDH assay components.25 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.26 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.,27 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.27
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.21,28 Similarly, Kroll et al.26 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.).29–31 The presence of serum in the experiment affects the result significantly. Kocbach et al.32 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.29 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.33 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.33 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, Co3O4 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).34 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.34 In a study by Kroll et al.,26 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 µg 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.35 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.
Another method to detect the free radical generation by ENMs is EPR (electroparamagnetic resonance), which with the use of specific spin traps or probes, and specific reagents could allow the quantification and identification of the type of free radical species generated. The potential interference in EPR-based methods in reactive oxygen species (ROS) measurement may be due to the interaction of ENMs with spin-trapping agents, which could ultimately alter the chemical and physical properties of ENMs. Such observations could provide the incorrect information about NP toxicity. Therefore, a careful check by a specific ROS donor system spiked with NPs should be used.36
Assessment of ROS production by formation of a nick in plasmid DNA due to the exposure of ENMs has been also reported.37,38 In this assay, the unwinding, nick or linearization of a coiled bacterial plasmid DNA is used to estimate free radical formation.39 This technique is only qualitative and not very sensitive. A potential interference for this method could be the binding of plasmid DNA at ENM surfaces, which may alter plasmid DNA mobility in electrophoresis.
1.3.6 Interference in Cellular Uptake Assays
Cellular internalization of ENMs is of central importance especially for biomedical applications such as targeted drug/gene delivery to fight complex diseases such as cancer.40 Also, during interpretation of the toxicity data it is essential to assess NP uptake in the cell and correlate it with the cellular responses. Transmission electron microscopy (TEM), scanning electron microscopy, backscattered electron energy-dispersive X-ray spectroscopy (SEM-BSE-EDS), confocal and fluorescence microscopy, reflection-based imaging, dark-field imaging and flow cytometry are the most common methods used to detect ENMs in cells.
Although these techniques have the advantage of tracking ENMs in the cell as well in cellular organelles, there are certain drawbacks. For example, in electron microscopy, the samples have to be fixed, hence, uptake in live cells cannot be monitored. A critical limitation is that TEM and SEM are operated under vacuum, so it is difficult to analyze liquid samples. Preparation steps of dehydration, cryofixation or embedding usually lead to sample alteration and dehydration artefacts. Another disadvantage of TEM is that the samples cannot be analyzed twice or used for validation of results. Further, the charging effects caused by the accumulation of static electric fields at the specimen due to the electron irradiation create confusion during imaging. Electron microscopy is also resource intensive and time consuming. Confocal and fluorescence microscopy, on the other hand, require probe tagging or fluorescence doping of ENMs for their detection. Because the ENMs are now modified, it is likely to alter their behaviour as well as their bioavailability. Currently, flow cytometry is being used to detect the internalization of ENMs in cells.41,42 The novelty in using flow cytometry lies in the fact that the uptake can be evaluated in live cells for several generations in real time. Flow cytometry also provides rapid, multiparametric, single-cell analysis with robust statistics (reduction of false-negative or type-II errors) due to the large number of events measured in three dimensions when compared with TEM. Despite having several advantages in detection of ENM uptake in cells, there are issues that need to be addressed in the use of flow cytometry.43 As the ENMs have very unique optical properties, scattering interference with the optical system of flow cytometry cannot be ruled out. Also, the size of the ENMs is very small, hence it is also difficult to interpret the influence on the scatter parameters of cells that represent ENM internalization or adsorption to the cell surface. It is also known that different ENMs have similar optical properties, hence the difference in the positioning of treated cells in dot-plots is difficult to analyze.
1.3.7 Interference with Cell Culture Media Components
The applications of ENMs require particles to be introduced into a living system (under in vitro or in vivo conditions). The bloodstream of an organism, cytoplasm of a cell and even the cell growing media, all are complex mixture of proteins, electrolytes, ions, nutrients, metabolites, etc. Upon exposure of ENMs to these aqueous media components, the physical and chemical interactions could affect the stability and properties of ENMs. Ag NPs have been reported to be oxidized under cell culture media, which led to a decrease in NP size and an increase in the Ag+ ion concentration in the media.44–47 Smaller Ag NPs have been shown to be rapidly internalized by cells, thereby causing toxicity. Similarly, Ag+ ions are also known to interact with cellular DNA and proteins, which led to the impaired functioning of cellular biochemical pathways. Additionally, spherical Au NPs with surface plasmon resonance (SPR) ∼520 nm have been shown to aggregate under cell culture media, which causes a shift in SPR to higher wavelengths (SPR ∼550–650 nm), which is the absorbance measurement point for most cell viability assays such as MTT, XTT and WST.47 Such events led to the increased absorbance, due to SPR of aggregated Au NPs and not due to cell viability, which causes misinterpretation of obtained cytotoxicity results. It has been shown that cell culture media supplemented with fetal bovine serum (FBS), upon interaction with ENMs, forms a protein corona over the NP surfaces.48 Such events led to the decrease in effective concentration of FBS available to NP-treated cells, which creates ambiguity in effects observed that may be due to NP stress or strain mediated by FBS deprivation.
1.3.8 Interference due to Oxidation State Change in Redox-active ENMs
ENMs are able to undergo oxidation/reduction reactions, which also governs the properties of NMs.19 The complex nature of the cytoplasmic environment may lead to an alteration in oxidation states, which could also affect cellular uptake and thus toxicity. For example, iron oxide NPs showed significant differences in cellular uptake and DNA damage depending on the oxidation state of iron (Fe2+ or Fe3+). It was observed that Fe3+ ions cause more genotoxicity than Fe2+ ions, which correlated well with cellular uptake.49 Therefore, cellular toxicity assessment of Fe2+ ions may show a toxic response due to their conversion to Fe3+ ions.50 Such redox-active NPs, which exhibit reactions in the cytoplasm may have their toxicity assessment misinterpreted. Therefore, strategies are needed to design ENPs that are chemically stable and oxidation resistant, without compromising on cellular damage. Similarly, cerium oxide NPs (nanoceria) also show oxidation-state-dependent properties. Nanoceria in its 3+ oxidation state shows superoxide dismutase-like properties,51 and in its 4+ oxidation state it shows catalase-like properties.52 The inter-conversion of oxidation state of nanoceria has been observed under physiological conditions of pH and buffer concentration.53
1.3.9 Misinterpretation of TEM Images
While TEM is a powerful instrument to image NMs made up of metals, oxides or semiconductors, there are some potential complications that may arise during imaging.
Particle agglomeration: TEM grid preparation involves solvent evaporation, which brings particles closer, thus particles look like aggregates. Therefore, it is not necessarily the case that the presence of aggregates viewed under TEM is symptomatic of aggregates in the starting suspension. Similarly, sections from cells/tissues also show black aggregates, which very frequently happen to be misinterpreted as NPs. The cytoplasm is full of small nanometre-size particulates, that may appear similar to NPs. The possible solution to this could be the use of more sophisticated instruments such as EELS (electron energy loss spectroscopy) or STEM (scanning transmission electron microscopy), which provide the in situ EDX (energy dispersive analysis of X-rays) analysis of the elemental composition of the material being imaged under TEM.
Impurities in the TEM sample: the principle behind TEM relies on differences in electron density between the sample and the surrounding matrix. Therefore, the presence of volatile organic molecules that have not been removed under vacuum, during sample preparation, or the presence of inorganic, electronically less dense, compounds gives poorer contrast between the sample and the surroundings, and compromises the image quality. This could possibly be avoided by the removal of impurities from the ENMs after synthesis by simple dialysis or centrifugation at high speed.
1.3.10 Interference with the Comet Assay
The comet assay is a simple, rapid and sensitive technique used to detect the single- and double-stranded DNA damage in individual cells (in vitro and in vivo). This is the most frequently used screening test for the quantification of alkali-labile sites, oxidative DNA damage, DNA–DNA or DNA–protein cross-linking and abasic-site DNA damage. The comet assay has also been used to detect damaged bases by incubating nucleoids with lesion-specific endonucleases, such as endonuclease III (Endo III) and FPG, which recognise oxidized pyrimidines and purines, respectively.
It is now well established, that ENMs can enter into the nucleus and can interact with the genome of the cell. Hence, in the comet assay where individual cells are analyzed, the probability of the presence of ENMs in the comet head (nucleoid) and their possibility to induce additional DNA damage during the assay cannot be ruled out (Stone et al.36 ).
The presence of ENMs closer to DNA also increases the probability of the interaction of ENMs with enzymes. It has recently been shown that the incubation of the ENMs and ions with FPG enzyme leads to the total loss of the ability of the enzyme to detect oxidatively damaged DNA in the comet assay (Kain et al.33 ). This disturbance is most likely due to the binding of ions to the –SH groups at the active site, or physical hindrance due to ENM binding, which prevents the enzyme action. This may result in the false interpretation of ENM properties. Also, the interference of ENMs with the staining process as well as the induction in fluorescence intensity due to autofluorescence of ENMs is also reported. It was also observed that ethidium bromide stained DNA/comet controls and TiO2 NP-treated cells faded after some time. However, there was some autofluorescence visible in the comet head of TiO2 NP-treated cells. The particles were evident when the same comet was exposed to bright light.
1.3.11 Interference in Micronucleus Assays
A micronucleus is a chromatin-containing structure in the cytoplasm, surrounded by a membrane without any detectable link to the cell nucleus. They are formed during the anaphase stage of cell division from the chromosomal fragments or whole chromosomes that are left behind when the nucleus divides. The micronucleus test is based on the scoring and comparison of the micronuclei in control and treated cells. This assay has been widely used to assess the genotoxic and carcinogenic potential of ENMs. As ENMs have the tendency to agglomerate, it has been observed that at higher concentrations ENMs are deposited on the cell surface/slide. The deposition of ENMs on the cell surfaces/slide during slide preparation hinders the counting of micronuclei thus the overall assay results.
In essence, the analysis of the literature and issues delineated in this chapter make it clear that before interpretation of observations on predicting the toxicity of the NPs, a meticulous identification and investigation of all the possible factors that interfere in toxicity assays is mandatory. It is therefore necessary to characterize NMs well in terms of size, activity and cross-reactivity with different biological macromolecules. The characterization of ENMs in terms of its shape, size, composition, and surface coating after dispersion in appropriate biologically relevant buffer, needs to be well understood during toxicity evaluation. Further, selection of a suitable test model system, the dose of exposure and appropriate assays are also critical factors to be considered for ENM toxicity investigation. The observations reported in the literature and described in this chapter stress the need to evaluate interferences between ENMs and assay components that may alter the actual interpretation of toxicity. Therefore, all toxicity methods should be further verified by alternative assays to confirm the accuracy of the experimental outcomes.
The advancements in nanotechnology will continue to push newer types of NMs in products that need toxicity data for sustainable applications. Therefore, new and improved throughput methods for NM toxicity assessment are required. A few methodologies based on predictive computational models,54,55 mechanism-centred high-throughput testing,55 genome arrays56 and high-throughput screening57 are being currently explored, but have not been introduced in toxicity assay mandates.
Despite these issues, nanotechnology remains a useful approach for the production of non-toxic, safe and highly effective medicines for treatment of deadly diseases at low doses and cost. The full potential of nanotechnology can only be realised with the collaborative efforts of experts, from nanoscience, physics, chemistry, biology, medicine and toxicology, to overcome hurdles associated with toxicity and safety assessment.
Funding received from the Department of Biotechnology, Government of India under the project “NanoToF: Toxicological evaluation and risk assessment on Nanomaterials in Food” (grant number BT/PR10414/PFN/20/961/2014) is gratefully acknowledged. Financial assistance by The Gujarat Institute for Chemical Technology (GICT) for the Establishment of a Facility for environmental risk assessment of chemicals and nanomaterials and Centre for nanotechnology research and applications (CENTRA) is also acknowledged.