A Review on Conventional and Advanced Methods for Nanotoxicology Evaluation of Engineered Nanomaterials
Abstract
:1. Introduction
2. Nanotoxicology and the Different Factors Influencing the Toxicity of Nanomaterials
3. Possible Mechanism of Nanomaterial Toxicity
4. The Different Approaches to Evaluate the Toxicity of Nanomaterials
4.1. Conventional Methods
4.1.1. In Vitro Methods
4.1.2. In Vivo Methods
4.2. Advanced Approaches to Assess Nanotoxicity
4.2.1. Atomic Force Microscopy (AFM)
4.2.2. Biomimetic 3-D Lung-on-a-Chip
4.2.3. Carbon Fiber Microelectrodes (CFMEs)
4.2.4. Fluidic-Based Cell-on-Chip (CoC)
4.2.5. High-Throughput Nanotoxicity Screening
4.2.6. Lateral Flow Immunoassay (LFIA)
4.2.7. Organ-on-Chip
4.2.8. Precision-Cut Tissue Slices
5. Toxicity of Carbon, Polymer, and Metallic-Based Nanocomposites and Their Toxicological Side Effects
5.1. Carbon Nanotube-Based Nanocomposites
5.2. Chitosan-Based Nanocomposites
5.3. Cyclodexdrin Nanosponge-Based Composites
5.4. Metallic-Based Nanoparticles
6. Challenges and Limitations
7. Conclusions and Recommendations
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Parameters | Tests | Description | References |
---|---|---|---|
Proliferation Assay | DNA Content | Proliferation can be quantified by observing and counting cells in mitosis while agents that hinder or cause mitotic progression are identified. Results are normally represented as a mitotic index (number of cells undergoing mitosis/total number of cells in a given population). Ki-67 nuclear antigen or a compound able to prevent cells in metaphase, such as colchicine, may be used. | [24] |
[3H] thymidine incorporation | This technique requires incubation (24–48 h) with [3H] thymidine. During the S phase, DNA of viable cells pick up [3H] thymidine, thus indicating the number of cells undergoing proliferation. | [35] | |
Bromodeoxyuridine (BrdU) incorporation | BrdU has enhanced specificity for cells going through DNA synthesis compared to [3H] thymidine, and a flow cytometry or special antibodies can be used to detect its presence. This assay can be used to investigate the proliferative as well as the antiproliferative effects of nanoparticles. | [24] | |
Ki-67 assay | Apart from G0, the nuclear antigen Ki-67 is present in all cell cycle steps and is detected through immunohistochemistry or spectrophotometrically. | [34,36] | |
Apoptosis Assay | Caspase assays | This assay is a luminescence-based test that measures caspase-7 and caspase-3 activities. Upon addition to caspase reagent to cells treated with NP, there is cell lysis followed by substrate cleavage using caspase, and then the luciferase produces a luminescent signal, which is comparable to the available quantity of caspase activity. | [37] |
TUNEL assay (Terminal deoxynucleotidyl transferase dUTP(deoxyuridinetriphoshate) nick end labelling) | It relies on the detection of DNA fragments produced in the last steps of apoptosis; the ends of DNA fragmented by endonucleases are labelled with biotinylated nucleotides conjugated to bromodeoxyuridine (BrdU), which can be identified by making use of a diaminobenzidine chromogen and streptavidin-horseradish peroxidase by fluorescent microscopy, or light microscopy or an immunohistochemical assay. | [38] | |
Annexin V | It is commonly utilized to identify apoptotic cells, which bind strongly to phosphatidylserine in a calcium-dependent manner. Phosphatidylserine is usually excluded from the plasma membrane’s extracellular surface but flips from the inner to the outer side upon the onset of apoptosis. The presence of phosphatidylserine on the extracellular surface sustains the membrane’s integrity while signaling for platelet aggregation and macrophage consumption. Calcium-mediated annexin V binds to the exposed phosphatidylserine as a shield from coagulation cascades to hinder rampant blood coagulation from the natural cell cycle. Therefore, the apoptotic cells can be detected using fluorescently labelled Annexin V. | [16,19] | |
Genotoxicity Assays | Ames assay | It is applied to test reverse mutation in Salmonella typhimurium. These bacteria have a mutation on the HIS operon and cannot generate the amino acid histidine, which is essential for bacterial replication. Exposing the bacteria to NP enables the bacteria to reverse the HIS operon’s mutation, resulting in histidine generation and colony development, which can be counted. Base-pair substitutions due to reverse mutations at the tryptophan locus can be tested in Escherichia coli (WP2uvrA). | [24,39] |
Comet Assay | It is used for quantifying and assessing DNA damage in cells. Cells embedded in a thin agarose gel are deposited on a microscope slide, and cellular proteins are obtained from the cells by lysing. The DNA is allowed to uncoil in neutral or alkaline conditions, and then it goes through electrophoresis, enabling the damaged DNA fragments to move away from the nucleus. The degree of fluorescence in the tail length, tail, and head is measured by staining with ethidium bromide or propidium iodide. The extent of DNA liberated from the comet’s head is immediately comparable to the amount of damaged DNA. | [40,41] | |
Measurement of oxidized guanine bases | Single base changes within a specific gene can be detected by assaying any one of several oxidized guanine bases, e.g., 7,8-dihydro-oxodeoxyguanine (oxo-dG) and 8-hydroxydeoxyguanosine (8-OHdG). The changes of these bases are frequently a result of oxidative damage and are measured through immunohistochemistry or HPLC. | [42] | |
Chromosomal aberration induction | This technique entails evaluating the influence of NPs on the number of cells and changes in chromosomes’ morphological appearance. Since cells are more sensitive during the S-phase, they are treated with NPs at this stage, followed by treatment at planned time intervals with colcemid or colchicine. Trypsinated cells are counted, and chromosomes are prepared and stained with Giemsa stain for microscopic evaluation of aberration (chromatid breaks, chromatid exchange, chromosome breaks, chromosome fragmentation). | [43] |
Organism | Description | References |
---|---|---|
Arabidopsis thaliana | It is a simple model system because its life cycle is rapid, it can easily be cultivated in a small space, and the information regarding its genome can also be clearly obtained. During the seedling period, A. thaliana is exposed to NPs for a given period of time; then, using the microarray technique, genomic analysis of harvested roots and leaves is done. | [52] |
Dapniapulex and Daphnia magna | They are small planktonic crustaceans primarily used for pre-screening since the biological differences between crustaceans and humans are extremely wide. They can be used to evaluate the effect of NP on mortality and reproduction. | [53] |
Caenorhabditis elegans | After exposing worms to NPs, a customized microfluidic chip is used to measure the level of metallothionein (mtl-2) gene expression, behavior, and worm length/width. Evaluating the physiological responses of C. elegans against chronic or acute anti-inflammatory and neurological therapeutics is helpful because the human biochemical pathways correlate well with the metabolic pathways and stress-associated markers of C. elegans. | [62] |
Drosophila melanogaster | Different modes of nanotoxicity like metabolic defects, fecundity, genotoxicity, and oxidative stress can be investigated in D. melanogaster. | [63] |
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Leudjo Taka, A.; Tata, C.M.; Klink, M.J.; Mbianda, X.Y.; Mtunzi, F.M.; Naidoo, E.B. A Review on Conventional and Advanced Methods for Nanotoxicology Evaluation of Engineered Nanomaterials. Molecules 2021, 26, 6536. https://doi.org/10.3390/molecules26216536
Leudjo Taka A, Tata CM, Klink MJ, Mbianda XY, Mtunzi FM, Naidoo EB. A Review on Conventional and Advanced Methods for Nanotoxicology Evaluation of Engineered Nanomaterials. Molecules. 2021; 26(21):6536. https://doi.org/10.3390/molecules26216536
Chicago/Turabian StyleLeudjo Taka, Anny, Charlotte Mungho Tata, Michael John Klink, Xavier Yangkou Mbianda, Fanyana Moses Mtunzi, and Eliazer Bobby Naidoo. 2021. "A Review on Conventional and Advanced Methods for Nanotoxicology Evaluation of Engineered Nanomaterials" Molecules 26, no. 21: 6536. https://doi.org/10.3390/molecules26216536