Factors affecting thein vitromicronucleus assay for evaluation of nanomaterials

Abstract

A number of in vitro methodologies have been used to assess the genotoxicity of different nanomaterials, including titanium dioxide nanoparticles (TiO NPs) and silver nanoparticles (AgNPs). The in vitro micronucleus assay is one of the most commonly used test methods for genotoxicity evaluation of nanomaterials. However, due to the novel features of nanomaterials, such as high adsorption capacity and fluorescence properties, there are unexpected interactions with experimental components and detection systems. In this study, we evaluate the interference by two nanoparticles, AgNPs and TiO NPs, with the in vitro micronucleus assay system and possible confounding factors affecting cytotoxicity and genotoxicity assessment of the nanomaterials including cell lines with different p53 status, nanoparticle coatings and fluorescence, cytochalasin B, fetal bovine serum in cell treatment medium and different measurement methodologies for detecting micronuclei. Our results showed that micronucleus induction by AgNPs was similar when evaluated using flow cytometry or microscope, whereas the induction by TiO NPs was different using the two methods due to TiO's fluorescence interference with the cytometry equipment. Cells with the mutated p53 gene were more sensitive to micronucleus induction by AgNPs than the p53 wild-type cells. The presence of serum during treatment increased the toxicity of AgNPs. The coatings of nanoparticles played an important role in the genotoxicity of AgNPs. These collective data highlight the importance of considering the unique properties of nanoparticles in assessing their genotoxicity using the in vitro micronucleus assay.

Micronuclei Detection by Flow Cytometry as a High-Throughput Approach for the Genotoxicity Testing of Nanomaterials

“…No MN induction was detected when NM100 and NM101 were tested in the CBMN assay, although a slight effect was observed for NM101 in one of the tested concentrations when FC was used, supporting the advantage of the MN scoring by the last methodology (results indicated in Table 2) [19]. Moreover, negative results were also obtained when human lymphoblastoid TK6 cells were exposed to different concentrations of TiO 2 NPs [20]. Nevertheless, both studies argued that the negative results were an artifact due to Nanomaterials 2019, 9, 1677 9 of 15 the interference of the TiO 2 fluorescence with the cytometry equipment.…”

“…Despite a large number of genotoxicity studies carried out with this nanomaterial, only a few of them used MN as a potential indicator of genotoxicity. From them, only two have recently used FC methodology to evaluate MN induction [19,20]. The evaluation of TiO 2 NPs in two different cell lines (BEAS-2B and HepG2) showed that positive effects were only observed when 10% of fetal bovine serum was used in the culture medium.…”

“…The evaluation of TiO2NPs in two different cell lines (BEAS-2B and HepG2) showed that positive effects were only observed when 10% of fetal bovine serum was used in the culture medium. From the two studies found in the literature evaluating MN induction by flow cytometry [19,20], only one used BEAS-2B cells and three different TiO 2 NPs (NM100, NM101 and NM103) at concentrations ranging from 1 to 30 µg/mL, two of them (NM100 and NM101) also used in our study. No MN induction was detected when NM100 and NM101 were tested in the CBMN assay, although a slight effect was observed for NM101 in one of the tested concentrations when FC was used, supporting the advantage of the MN scoring by the last methodology (results indicated in Table 2) [19].…”

“…An updated revision of the original method discussing advantages, limitations and future recommendations have been carried out [18]. The FCMN assay has already been used to test several NMs such as TiO 2 NPs [19,20], AgNPs [20][21][22][23] and SiO 2 NPs [24].…”

The size‐dependent apoptotic effect of titanium dioxide nanoparticles on endothelial cells by the intracellular pathway

“…29 It is now known that the intracellular approach of apoptosis is mainly initiated by the induction of p53 as a response to DNA damage induced by genotoxic agents. 29 A number of studies have shown that nano-TiO 2 can induce DNA damage and adversely affect both major DNA repair mechanisms: base excision repair and nucleotide excision repair, showing a genotoxic potential to human cells 10,31,[42][43][44][45] and animal models. 24,46,47 We also found that the four sizes of nano-TiO 2 could induce significant DNA damages and micronuclei in human BEAS-2B cells (unpublished data).…”