Suitability of the In Vitro Cytokinesis-Block Micronucleus Test for Genotoxicity Assessment of TiO2 Nanoparticles on SH-SY5Y Cells

Abstract

Standard toxicity tests might not be fully adequate for evaluating nanomaterials since their unique features are also responsible for unexpected interactions. The in vitro cytokinesis-block micronucleus (CBMN) test is recommended for genotoxicity testing, but cytochalasin-B (Cyt-B) may interfere with nanoparticles (NP), leading to inaccurate results. Our objective was to determine whether Cyt-B could interfere with MN induction by TiO2 NP in human SH-SY5Y cells, as assessed by CBMN test. Cells were treated for 6 or 24 h, according to three treatment options: co-treatment with Cyt-B, post-treatment, and delayed co-treatment. Influence of Cyt-B on TiO2 NP cellular uptake and MN induction as evaluated by flow cytometry (FCMN) were also assessed. TiO2 NP were significantly internalized by cells, both in the absence and presence of Cyt-B, indicating that this chemical does not interfere with NP uptake. Dose-dependent increases in MN rates were observed in CBMN test after co-treatment. However, FCMN assay only showed a positive response when Cyt-B was added simultaneously with TiO2 NP, suggesting that Cyt-B might alter CBMN assay results. No differences were observed in the comparisons between the treatment options assessed, suggesting they are not adequate alternatives to avoid Cyt-B interference in the specific conditions tested.

Adapting the in vitro micronucleus assay (OECD Test Guideline No. 487) for testing of manufactured nanomaterials: recommendations for best practices

“…Cytochalasin B has been shown to interfere with actin filaments required for endocytosis and thus co-exposures should be avoided; this recommendation have been extensively outlined in previous publications [ 1 , 5 , 42 ]. Recently, Fernandez-Bertolez (2022) have reported that cellular uptake of TiO 2 ENPs and micronucleus induction is not affected by the presence or absence of cyto B during the exposure period in SH-SY5Y cells [ 43 ]. This could indicate that in some cells other modes of nanomaterial uptake is present.…”

Editorial of Special Issue: The Toxicity of Nanomaterials and Legacy Contaminants: Risks to the Environment and Human Health

“…In this context, and considering the existing evidence that cytochalasin-B, used in in vitro cytokinesis-block micronucleus (CBMN), affects the uptake of nanomaterials, hindering its use in the genotoxicity testing of nanomaterials. Fernández-Bertólez et al [20] tested TiO 2 NP uptake and induced genotoxicity in neuroblastoma (SH-SY5Y) cells using flow cytometry and CBMN, after three different treatment options with cytochalasin-B, as recommended by the Organisation for Economic Co-operation and Development (OECD). The results showed that, even though there was no interference of cytochalasin-B in the uptake, micronuclei induction was affected by the presence of cytochalasin-B in the medium, possibly causing false positive results.…”