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

Natalia Fernández-Bertólez,Fátima Brandão,Blanca Laffon,Carla Costa,João Paulo Teixeira,Eduardo Pásaro,Vanessa Valdiglesias

doi:10.3390/ijms22168558

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.

Highlights

This article is an open access articleThe rapid development of nanotechnology has brought huge benefits to our daily lives, but it can entail potential threats to human health and environment [1,2]
Our results showed no differences between post-treatment and co-treatment, and only a slight decrease in MN frequency in delayed co-treatment, limited to the highest concentration and the longest treatment tested, with regard to the other two treatment options
Despite previous studies highlighted the possible interference of Cyt-B on NP cellular uptake and, on MN detection by cytokinesis-block micronucleus (CBMN) test, current data do not support that hypothesis in the case of SH-SY5Y cells exposed to TiO2 NP

Summary

Introduction

This article is an open access articleThe rapid development of nanotechnology has brought huge benefits to our daily lives, but it can entail potential threats to human health and environment [1,2]. The in vitro mammalian cell micronucleus (MN) test is one of the assays recommended by the Organisation for Economic Cooperation and Development (OECD) for genotoxicity testing of chemicals (Test Guideline (TG) 487) [13], and is considered to be a well-established, reliable, accurate and reproducible endpoint in genotoxicity evaluation [14,15,16]. This test method has been endorsed as reliable and relevant by the European Centre for the Validation of Alternative Methods (ECVAM) Scientific Advisory

Objectives

Methods

Results

Discussion

Conclusion