Adaptations of the in vitro MN assay for the genotoxicity assessment of nanomaterials

Abstract

The issue of appropriate testing strategies has been raised for the genotoxicity assessment of nanomaterials. Recently, efforts have been made to evaluate the adequacy of Organisation for Economic Co-operation and Development-standardised tests to assess the genotoxicity of nanomaterials. The aim of this review was to examine whether the current guideline for the in vitro micronucleus (MN) assay is applicable for testing nanomaterials. From a Pubmed literature search, 21 available studies were identified for analysis. We reviewed all protocols used for testing nanomaterials with the in vitro MN assay. All studies were categorised based on the particle type and size. Different aspects of the protocols were evaluated such as the exposure (duration and doses), the cytochalasin-B treatment, serum levels and cytotoxicity assessment. Sixteen of the 21 studies demonstrated increased frequencies of MN. Some recommendations regarding the protocol were formulated to maximise sensitivity and avoid false negatives. Determination of the cellular dose was advised for a better interpretation of MN frequency results. The level of serum can modulate the cellular response, therefore the serum percentage used should enable cell growth and proliferation and a maximal sensitivity of the assay. Furthermore, different types of cytochalasin-B treatment were used, co-treatment, post-treatment and delayed co-treatment. In order to avoid decreased cellular uptake as a consequence of actin inhibition, post-treatment or delayed co-treatment is suggested. Exposure during mitosis should be recommended to allow contact with the chromatin or mitotic apparatus for nanomaterials that are unable to cross the nuclear membrane. With these adaptations, the in vitro MN assay can be recommended for genotoxicity testing of nanomaterials.

Assessment of the genotoxic potential of two zinc oxide sources (amorphous and nanoparticles) using the in vitro micronucleus test and the in vivo wing somatic mutation and recombination test

“…According to our results (Fig. 3andTable 1), ZnO NPs were cytotoxic at concentrations equal to or higher than 240.0 µM and genotoxic (clastogenic or aneugenic) only at 120.0 µM Gonzalez et al (2011) andDoak et al (2012). discussed that methodological variations in in vitro micronucleus test as described in the draft OECD guideline(OECD 487, 2010) can influence in the genotoxicity testing of nanomaterials.…”

Genotoxicity testing of PLGA–PEO nanoparticles in TK6 cells by the comet assay and the cytokinesis-block micronucleus assay

“…Cytochalasin B also inhibits endocytosis, which is an important cellular uptake mechanism favoured by some nanomaterials. For the MN assay with nanomaterials one should consider actin-dependent uptake when cytochalasin B is used [27]. To avoid the interference of cytochalasin B with nanoparticle treatment and to assess the most appropriate and effective time point of inclusion of cytochalasin B, two different experimental protocols have been used in our study: the addition of cytochalasin B during the final 24 h of treatment, but with the nanomaterial present for the duration of the cytochalasin B treatment (1st protocol); and the addition of cytochalasin B after removal of the nanomaterial medium (2nd protocol).…”

“…When aneugenic effects are suspected, as shown by Elhajouji et al [15] and as recommended by the draft OECD guidelines [11], the analysis of MN in mononucleated cells may be an additional parameter in the CBMN assay. Indeed, aneugenic compounds may induce mitotic slippage resulting in an increase of mononucleated cells with MN and almost no increase of MN in binucleated cells [27]. Rosefort et al [28] tested some typical aneugens and clastogens in cultures of whole blood lymphocytes from four donors with the CBMN assay.…”

INDUCCIÓN DE MICRONÚCLEOS EN CÉLULAS MERISTEMÁTICAS DE LA RAÍZ DE Vicia faba TRATADAS CON DIFERENTES CONCENTRACIONES DE MARVEL®

“…La inducción de ICH y la alteración tanto en la progresión del ciclo celular y los IM se ha encontrado en linfocitos humanos in vitro y células de ovario de hámster Chino (CHO-K1) (González et al 2006;. Además, se informó que el herbicida induce roturas de hebra de ADN reveladas por el ensayo cometa (González et al 2007), así como MN en células de CHO-K1 (González et al 2011). Por último, también se reportaron resultados positivos en la aplicación del ensayo de MN en Tradescantia (Mohammed y Ma 1999).…”