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of innate immune system responsible for enhancing the ability of antibodies and
phagocytic cells to clear pathogens from an organism, and its in vitro evaluation is
strongly dependent on the applied matrix (Neun and Dobrovolskaia 2011).
Thrombogenicity is a complex process encompassing multiple cell types (thrombocytes, leucocytes, endothelial cells) and plasma coagulation factors; therefore, a
multiple-assay approach is highly recommended (Myerson et al. 2011).
8.4.4 Genotoxicity
Genotoxicity is considered a fundamental in vitro endpoint in nanotoxicology, due
to its correlation with cancer risk (Doktorovova et al. 2014a, c; Souto et al. 2020d).
There is no single test established to detect all genotoxins, thus when performing
genotoxicity assays, a multiple approach should be carried out. However, within the
selection of a suitable test battery, critical endpoints should be considered, namely,
gene mutation, structural and numerical chromosome aberrations (Bastús and
Puntes 2017), and generally, those endpoints can be assessed by the following in
vitro assays: (i) in vitro mammalian cell gene mutation test; (ii) in vitro mammalian
chromosome aberration test; and (iii) in vitro mammalian cell micronucleus test
(Rasmussen et al. 2016). The first assay, cell gene mutation test, is usually used to
detect gene mutations at hypoxanthine-guanine phosphoribosyl transferase (HPRT),
and at a transgene of xanthine-guanine phosphoribosyl transferase (XPRT). The
HPRT and XPRT genes mutation tests detect different spectra of genetic events. The
chromosome aberration test identifies structural chromosome aberrations—in chromosome or chromatid—caused by chemicals and other xenobiotics. After treatment, stained metaphase cells are analyzed microscopically for the presence of
aberrations. Lastly, the micronucleus test can detect the presence of micronuclei in
cytoplasm of interphase cells originating from acentric chromosome fragments or
whole chromosomes unable to migrate during cell division (Rasmussen et al. 2016;
Bastús and Puntes 2017). This chromosomal alteration concerns cells undergoing
mitosis, and the use of cytochalasin B as cytokinesis blocker permits the analysis of
those cells that have completed mitosis (Manshian et al. 2015; Branica et al. 2016).
On the other hand, as a simpler alternative DNA damage testing can also be considered for genotoxicity assessment. To evaluate DNA fragmentation, the marker of
DNA damage, two different in vitro assays are reported: comet assay and TUNEL
assay (Silva et al. 2019a). Comet assay is based on identification and quantification
of comet tails after gel electrophoresis of lysed cells, and the brighter and longer the
tail, the higher the level of damage is (Love et al. 2012; Stone et al. 2009; Burlinson
2012). TUNEL assay consists in a method for detecting apoptotic DNA fragmentation relying on the binding of terminal deoxynucleotidyl transferase (TdT), tagged
with a fluorochrome or another marker, to 3′-hydroxyl termini of DNA doublestrand breaks, thus detecting damaged cells (Love et al. 2012; Caballero-Diaz and
Cases 2016).
M. C. Teixeira et al.
of innate immune system responsible for enhancing the ability of antibodies and
phagocytic cells to clear pathogens from an organism, and its in vitro evaluation is
strongly dependent on the applied matrix (Neun and Dobrovolskaia 2011).
Thrombogenicity is a complex process encompassing multiple cell types (thrombocytes, leucocytes, endothelial cells) and plasma coagulation factors; therefore, a
multiple-assay approach is highly recommended (Myerson et al. 2011).
8.4.4 Genotoxicity
Genotoxicity is considered a fundamental in vitro endpoint in nanotoxicology, due
to its correlation with cancer risk (Doktorovova et al. 2014a, c; Souto et al. 2020d).
There is no single test established to detect all genotoxins, thus when performing
genotoxicity assays, a multiple approach should be carried out. However, within the
selection of a suitable test battery, critical endpoints should be considered, namely,
gene mutation, structural and numerical chromosome aberrations (Bastús and
Puntes 2017), and generally, those endpoints can be assessed by the following in
vitro assays: (i) in vitro mammalian cell gene mutation test; (ii) in vitro mammalian
chromosome aberration test; and (iii) in vitro mammalian cell micronucleus test
(Rasmussen et al. 2016). The first assay, cell gene mutation test, is usually used to
detect gene mutations at hypoxanthine-guanine phosphoribosyl transferase (HPRT),
and at a transgene of xanthine-guanine phosphoribosyl transferase (XPRT). The
HPRT and XPRT genes mutation tests detect different spectra of genetic events. The
chromosome aberration test identifies structural chromosome aberrations—in chromosome or chromatid—caused by chemicals and other xenobiotics. After treatment, stained metaphase cells are analyzed microscopically for the presence of
aberrations. Lastly, the micronucleus test can detect the presence of micronuclei in
cytoplasm of interphase cells originating from acentric chromosome fragments or
whole chromosomes unable to migrate during cell division (Rasmussen et al. 2016;
Bastús and Puntes 2017). This chromosomal alteration concerns cells undergoing
mitosis, and the use of cytochalasin B as cytokinesis blocker permits the analysis of
those cells that have completed mitosis (Manshian et al. 2015; Branica et al. 2016).
On the other hand, as a simpler alternative DNA damage testing can also be considered for genotoxicity assessment. To evaluate DNA fragmentation, the marker of
DNA damage, two different in vitro assays are reported: comet assay and TUNEL
assay (Silva et al. 2019a). Comet assay is based on identification and quantification
of comet tails after gel electrophoresis of lysed cells, and the brighter and longer the
tail, the higher the level of damage is (Love et al. 2012; Stone et al. 2009; Burlinson
2012). TUNEL assay consists in a method for detecting apoptotic DNA fragmentation relying on the binding of terminal deoxynucleotidyl transferase (TdT), tagged
with a fluorochrome or another marker, to 3′-hydroxyl termini of DNA doublestrand breaks, thus detecting damaged cells (Love et al. 2012; Caballero-Diaz and
Cases 2016).
M. C. Teixeira et al.