Table 1.

In vitro assays for evaluating nanotoxicity.

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]