Abstract
The micronucleus assay is an important technique used to evaluate genotoxic damage of chemical or physical agents (as ionizing radiations) on cells, based on quantification of cells bearing micronuclei, which are fragments derived from damage (breakage) of the DNA. Currently, this technique was updated to an automated approach that relies on plasma membrane dissolution to analyze fluorescent dye-labelled nuclei and micronuclei by flow cytometry. Cell suspensions were irradiated in PBS by a 60Co source in doses between 0 and 16 Gy, and incubated by 72h. Cell membranes were lysed in the presence of SYTOX Green and EMA dyes, so EMA-stained nuclei could be discriminated as from dead cells, and nuclei and micronuclei could be quantified. Amounts of micronuclei (percent of events) in the samples, were found to be proportional to radiation doses, and could be fitted to a linear-quadratic model (R² = 0.993). Only higher doses (8 and 16 Gy) and positive control could induce relevant increases in micronucleus amounts. The incorporation EMA showed an increase in irradiated cells. Mid- to high doses (4, 8 and 16 Gy) induced reduction of cell proliferation. Experiments showed the suitability of the technique to replace traditional microscopy analysis in evaluation of the effects of ionizing radiations on cells, with possibility to use in biological dosimetry.
Highlights
The in vitro micronucleus frequency test (FMN) is one of the methodologies of choice in the development of toxicological safety tests
The technique is based on the observation of accumulation of cytoplasmic micronuclei by blocking cytokinesis (CBMN), and is performed using cytochalasin B, a compound of fungal origin that can inhibit cytokinesis without inhibiting karyokinesis of the cell nucleus after DNA duplication
The technique is characterized as an in vitro test, and is a relatively fast and very efficient test system used to evaluate chemicals that can induce genotoxic damage, leading to the formation of micronuclei in the cytoplasm of cells. These micronuclei may originate from acentric fragments or whole chromosomes that are unable to migrate with the rest of the chromosomes during the anaphase of cell division [2]
Summary
The in vitro micronucleus frequency test (FMN) is one of the methodologies of choice in the development of toxicological safety tests. The technique is based on the observation of accumulation of cytoplasmic micronuclei by blocking cytokinesis (CBMN), and is performed using cytochalasin B, a compound of fungal origin that can inhibit cytokinesis (cell division itself) without inhibiting karyokinesis of the cell nucleus after DNA duplication In this way, cells treated by this compound exhibit two nuclear bodies, being called binucleate cells. The technique is characterized as an in vitro test, and is a relatively fast and very efficient test system used to evaluate chemicals that can induce genotoxic damage, leading to the formation of micronuclei in the cytoplasm of cells These micronuclei may originate from acentric fragments or whole chromosomes that are unable to migrate with the rest of the chromosomes during the anaphase of cell division [2]. The present work used a flow cytometry testing protocol to assess genotoxic damage caused by 60Co on CHO-KI cells
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