Cytokinesis-Block Micronucleus Cytome Assay Evolution into a More Comprehensive Method to Measure Chromosomal Instability

Abstract

This review describes the cytokinesis-block micronucleus (CBMN) cytome assay and its evolution into a molecular cytogenetic method of chromosomal instability (CIN). Micronuclei (MNi) originate from whole chromosomes or chromosome fragments that fail to segregate to the poles of the cell during mitosis. These lagging chromosomes are excluded from the daughter nuclei and are enveloped in their own membrane to form MNi. The CBMN assay was developed to allow MNi to be scored exclusively in once-divided binucleated cells, which enables accurate measurement of chromosome breakage or loss without confounding by non-dividing cells that cannot express MNi. The CBMN assay can be applied to cell lines in vitro and cells such as lymphocytes that can be stimulated to divide ex vivo. In the CBMN assay, other CIN biomarkers such as nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) are also measured. Use of centromere, telomere, and chromosome painting probes provides further insights into the mechanisms through which MNi, NPBs and NBUDs originate. Measurement of MNi is also important because entrapment within a micronucleus may cause chromosomes to shatter and, after nuclear reintegration, become rearranged. Additionally, leakage of DNA from MNi can stimulate inflammation via the cyclic GMP-AMP Synthase—Stimulator of Interferon Genes (cGAS-STING) DNA sensing mechanism of the innate immune system.

Acute Toxicity and DNA Instability Induced by Exposure to Low Doses of Triclosan and Phthalate DEHP, and Their Combinations, in vitro

“…We also observed that exposure to the both EDCs was able to impact the NDI of HepG2 cells and previous studies showed that oxidative damage in DNA is related to cell cycle arrest (Droge, 2002;Murray and Carr, 2018). Reduced NDI is associated with more mononucleated cells, when compared to bi-, tri-, and polynucleated ones, giving evidence of cell cycle arrest (Fenech, 2007(Fenech, , 2020. Interestingly, the most pronounced effects on NDI were seen when the cells were treated with the combinations of TCS and DEHP, at all doses.…”

“…In this context, MNs may also arise through gene amplification through fusion bridge breaking (BFB) cycles. In the case of NBUDs, DNA is selectively located at specific locations on the periphery of the nucleus and can be eliminated through nuclear sprouting, during S phase of the cell cycle; then, as consequence, extrusion of amplified DNA via nuclear sprouting may result in MN formations (Fenech and Crott, 2002;Mateuca et al, 2006;Fenech, 2020). It is also important to mention that we did not observed increase of NPBs in cells treated with TCS and DEHP.…”

Biomonitoring findings for occupational lead exposure in battery and ceramic tile workers using biochemical markers, alkaline comet assay, and micronucleus test coupled with fluorescence in situ hybridisation

“…We found no significant difference in NB formation between the exposed workers and controls (Table 3). This could be expected, considering how NBs as indicators of genetic instability are formed (94)(95)(96). They are the consequence of elimination of amplified genes and unresolved DNA repair complexes (94).…”

“…This could be expected, considering how NBs as indicators of genetic instability are formed ( 94 , 95 , 96 ). They are the consequence of elimination of amplified genes and unresolved DNA repair complexes ( 94 ). Inorganic lead did not activate any detoxification pathway or induce an over-expression of genes and consequently their copy number.…”

Inter-laboratory automation of thein vitromicronucleus assay using imaging flow cytometry and deep learning

“…Harmonised assay protocols and scoring approaches have been detailed by Organisation for Economic Cooperation and Development (OECD)-Test Guideline 487 (OECD 2016). In addition to regulatory compound screening, the assay is also widely used for more specific research and clinical purposes including compound mode-of-action determinations, tumour radiosensitivity prediction and inter-individual monitoring of lifestyle, occupational and environmental factors including radiation biodosimetry assessments (Decordier and Kirsch-Volders 2006;Fenech 2000;Fenech 2020;Kirsch-Volders et al 2011;Wang et al 2019).…”