Nanomaterial genotoxicity evaluation using the high-throughput p53-binding protein 1 (53BP1) assay

Abstract

Toxicity evaluation of engineered nanomaterials is challenging due to the ever increasing number of materials and because nanomaterials (NMs) frequently interfere with commonly used assays. Hence, there is a need for robust, high-throughput assays with which to assess their hazard potential. The present study aimed at evaluating the applicability of a genotoxicity assay based on the immunostaining and foci counting of the DNA repair protein 53BP1 (p53-binding protein 1), in a high-throughput format, for NM genotoxicity assessment. For benchmarking purposes, we first applied the assay to a set of eight known genotoxic agents, as well as X-ray irradiation (1 Gy). Then, a panel of NMs and nanobiomaterials (NBMs) was evaluated with respect to their impact on cell viability and genotoxicity, and to their potential to induce reactive oxygen species (ROS) production. The genotoxicity recorded using the 53BP1 assay was confirmed using the micronucleus assay, also scored via automated (high-throughput) microscopy. The 53BP1 assay successfully identified genotoxic compounds on the HCT116 human intestinal cell line. None of the tested NMs showed any genotoxicity using the 53BP1 assay, except the positive control consisting in (CoO)(NiO) NMs, while only TiO2 NMs showed positive outcome in the micronucleus assay. Only Fe3O4 NMs caused significant elevation of ROS, not correlated to DNA damage. Therefore, owing to its adequate predictivity of the genotoxicity of most of the tested benchmark substance and its ease of implementation in a high throughput format, the 53BP1 assay could be proposed as a complementary high-throughput screening genotoxicity assay, in the context of the development of New Approach Methodologies.

Iron Oxide (Magnetite)-Based Nanobiomaterial with Medical Applications—Environmental Hazard Assessment Using Terrestrial Model Species

“…Studies on NBMs’ toxicity, including Fe 3 O 4 PEG-PLGA, are mostly available for in vitro models, without much information on their effects on environmental species. Several in vitro cytotoxicity studies with Fe 3 O 4 PEG-PLGA indicate an overall low-to-no toxicity to different cell models [ 16 , 17 , 18 ]. Genotoxicity was also not observed in HCT116 cells exposed to Fe 3 O 4 PEG-PLGA up to 50 μg/mL [ 16 , 18 ], despite the increase in reactive oxygen species (ROS) levels observed above 10 μg/mL [ 16 ].…”

“…Several in vitro cytotoxicity studies with Fe 3 O 4 PEG-PLGA indicate an overall low-to-no toxicity to different cell models [ 16 , 17 , 18 ]. Genotoxicity was also not observed in HCT116 cells exposed to Fe 3 O 4 PEG-PLGA up to 50 μg/mL [ 16 , 18 ], despite the increase in reactive oxygen species (ROS) levels observed above 10 μg/mL [ 16 ]. In a study performed in an environmental species, i.e., using the fish cell lines RTL-W1 (CRL-2301, derived from the liver), RTgill-W1 (CRL-2523, derived from the branchial arc), and RTS-11 (derived from the spleen), up to 100 μg/mL within 24 h [ 19 ] of Fe 3 O 4 PEG-PLGA was also not acutely toxic.…”