The High-Throughput In Vitro CometChip Assay for the Analysis of Metal Oxide Nanomaterial Induced DNA Damage

Abstract

Metal oxide nanomaterials (MONMs) are among the most highly utilized classes of nanomaterials worldwide, though their potential to induce DNA damage in living organisms is known. High-throughput in vitro assays have the potential to greatly expedite analysis and understanding of MONM induced toxicity while minimizing the overall use of animals. In this study, the high-throughput CometChip assay was used to assess the in vitro genotoxic potential of pristine copper oxide (CuO), zinc oxide (ZnO), and titanium dioxide (TiO2) MONMs and microparticles (MPs), as well as five coated/surface-modified TiO2 NPs and zinc (II) chloride (ZnCl2) and copper (II) chloride (CuCl2) after 2–4 h of exposure. The CuO NPs, ZnO NPs and MPs, and ZnCl2 exposures induced dose- and time-dependent increases in DNA damage at both timepoints. TiO2 NPs surface coated with silica or silica–alumina and one pristine TiO2 NP of rutile crystal structure also induced subtle dose-dependent DNA damage. Concentration modelling at both post-exposure timepoints highlighted the contribution of the dissolved species to the response of ZnO, and the role of the nanoparticle fraction for CuO mediated genotoxicity, showing the differential impact that particle and dissolved fractions can have on genotoxicity induced by MONMs. The results imply that solubility alone may be insufficient to explain the biological behaviour of MONMs.

Dissolution Behaviour of Metal-Oxide Nanomaterials in Various Biological Media

“…For the hazard assessment, this indicates that the focus should be on dissolved species rather than particles in the case of this ENM. This observation is relevant for the interpretation of toxicity results as increased ENM solubility also affects toxicity [ 62 , 63 , 64 ]. While less pronounced, this effect of increased % solubility with a decreasing initial concentration was also observed for MnO 2 , CeO 2 , and Al 2 O 3 ENM in DMEM ( Figure 2 ).…”

Novel In Vivo CometChip Reveals NDMA-Induced DNA Damage and Repair in Multiple Mouse Tissues

“…Since then, the method has been demonstrated to be effective in more than three dozen publications. (Recent publications include: [ 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 ]). When cells are exposed to methylating agents, DNA strand breaks and alkali-labile sites can arise as transient intermediates during base excision repair (BER).…”

Toxicity of Metal Oxide Nanoparticles: Looking through the Lens of Toxicogenomics

“…For CuO, ZnO, and TiO 2 NPs, transmission electron microscopic (TEM) imaging was previously conducted [ 33 , 34 ], and the results showed their measured particle sizes to be <50 nm. The TEM sizing of both NiO and Al 2 O 3 NPs also showed mean measured particle sizes <50 nm ( Table 1 , Figure S1 ).…”

“…Recently, using in vitro global transcriptional analysis and cytotoxicity monitoring, Boyadzhiev et al, 2021 demonstrated that, for CuO NPs showing moderate solubility in cell culture medium, the toxicity is potentiated by the particle size, even though apical and molecular responses may be qualitatively similar between NP and dissolved Cu exposures [ 33 ]. Furthermore, the same authors recently examined the DNA damage potential of ZnO, CuO, and TiO 2 NPs, associated microparticles (MPs), and metal chloride salts and showed that ZnO NPs and MPs behaved similarly to dissolved Zn with respect to DNA damage induction, whereas significant differences were observed between CuO forms, with only NPs inducing DNA damage compared to MPs or dissolved Cu [ 34 ]. For the TiO 2 NPs of negligible solubility tested, silica and silica-alumina coatings and a rutile crystal structure potentiated a muted DNA damage response.…”