From the Cover: Does the Assessment of Nondisjunction Provide a More Sensitive Assay for the Detection of Aneugens?

TOXICOLOGICAL SCIENCES, 157(1), 2017, 20–29 doi: 10.1093/toxsci/kfx014 Advance Access Publication Date: January 14, 2017 Research article Does the Assessment of Nondisjunction Provide a More Sensitive Assay for the Detection of Aneugens? Joanne M. Elloway,*,1,4 Alexandra K. Davies,*,1,2,3 Julie E. Hayes,† and Ann T. Doherty* *AstraZeneca, Innovative Medicines and Early Development, Drug Safety and Metabolism (Discovery Safety), Unit 310—Darwin Building, Cambridge CB4 0WG, UK; and †AstraZeneca, Innovative Medicines and Early Development, Drug Safety and Metabolism (Discovery Safety), AstraZeneca R&D, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK 1 Joint first Authors (in this order in publication). 2 Present address: Department of Clinical Biochemistry, Cambridge Institute for Medical Research, University of Cambridge, Hills Road, Cambridge, UK. 3 Work conducted during industrial placement year at AstraZeneca. 4 To whom correspondence should be addressed at AstraZeneca, Innovative Medicines and Early Development, Drug Safety and Metabolism (Discovery Safety), Unit 310—Darwin Building, Cambridge Science Park Milton Road, Cambridge CB4 0WG, UK. Fax: þ44-1223223429. E-mail: . ABSTRACT The detection of aneugenic chemicals is important due to the implications of aneuploidy for human health. Aneuploidy can result from chromosome loss or nondisjunction due to chromosome mis-segregation at anaphase. Frequently, aneugens are detected using the in vitro micronucleus assay (IVM), with either centromere or kinetochore labeling. However, this method does not consider nondisjunction, the suggested predominant mechanism of spindle poison induced aneugenicity in primary human lymphocytes. Therefore, the IVM may be relatively insensitive in detecting aneuploidy. To investigate whether chromosome distribution analysis, specifically of nondisjunction, using chromosome-specific centromeric probes provides a more sensitive assay for aneugen detection, six reference aneugens with differing modes of action were tested on human lymphoblastoid TK6 cells. The results show that chromosome loss is a substantial part of the process leading to aneuploidy in TK6 cells. This differs from previous studies on human lymphocytes where nondisjunction has been described as the major mechanism of aneugenicity. However, in the current study more cells and types of aneugenic damage were analyzed. Although compound specific effects on nondisjunction were identified, chromosome distribution analysis did not provide increased sensitivity for the detection of aneugens: For the six reference aneugens examined, chromosome loss was shown at the same concentrations or lower than nondisjunction, even when nondisjunction levels were comparatively high. Therefore, in TK6 cells methods that detect chromosome loss, eg, the IVM, provide a more sensitive technique for the detection of aneugens than the measurement of nondisjunction. Key words: aneugen; nondisjunction; micronuclei. Aneugenicity is an umbrella term for multiple molecular mechanisms that involve compounds that act on any component of the cell division apparatus. Therefore, aneugens are a diverse class of chemicals. The ability to detect aneugenic chemicals is important due to the negative implications of aneuploidy on human health including congenital abnormalities and somatic cell disorders. Aneuploidy is also strongly correlated with tumor malignancy via the induction of chromosomal and genomic instability. A recent publication has shown the aneuploidy may also have a role in the initiation of carcinogenesis by the altering the expression of proteins related to DNA replication, in particular the reduction in expression of helicase MCM2-7(Passerini et al., 2016). C The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. V All rights reserved. For Permissions, please e-mail: 20 ELLOWAY ET AL. | 21 FIG. 1. Diagrammatic representation of the use of FISH techniques for distinguishing aneugenic and clastogenic events in binucleates. A pan-centromeric probe (denoted by green circles) can be used to identify micronuclei containing whole chromosomes. Chromosome-specific probes (red and green rectangles) can be used analyze chromosome distribution and identify nondisjunction. Aneuploidy can result from any compound that disrupts the cell division apparatus and manifests via two main processes. The first process is nondisjunction of chromosomes at anaphase, eg, through inappropriate attachment of the mitotic spindle resulting in the production of a 3:1 or 4:0 distribution of the chromosomes between the two daughter nuclei. The second process is chromosome loss, occurring if a chromosome lags at the metaphase plate due to misattachment of the spindle and is not incorporated into either daughter nuclei. The lost chromosome may be retained as a micronucleus, reincorporated into either daughter nuclei or lost from the cell (Parry et al., 2002). The most commonly used technique to investigate the aneugenic potential of compounds is the in vitro micronucleus assay (IVM) in combination with either centromere or kinetochore labeling. Micronuclei form in dividing cells from either acentric chromosome fragments (clastogenic events) or from whole chromosomes lost during telophase (aneugenic events), which are then enveloped in a nuclear membrane to give rise to an extranuclear body (Fenech, 2000). The expression of micronuclei is dependent upon cell division. Therefore, the identification of cells that have divided at least once during or after treatment is important. These can be identified through the use of a modification of the IVM which utilizes cytochalasin-B, an inhibitor of cytokinesis, leading to the formation of binucleated cells post mitosis (Fenech and Morley, 1985). There are several methods available to discriminate micronuclei induced by clastogenic and aneugenic events. These include the use of autoantibodies against kinetochores from patients with CREST syndrome (Moroi et al., 1980), antibodies against human centromeric proteins, and fluorescence in situ hybridization (FISH) using an alphoid DNA probe p82H specific to a centromeric sequence found on all human chromosomes (Mitchell et al., 1985). These methods discriminate clastogens and aneugens based on the proportion of induced micronuclei containing centromeric DNA or kinetochore protein; aneugens produce mainly centromere/ kinetochore-positive micronuclei (Cþ ve MN) and clastogens mainly centromere/kinetochore-negative C ve MN (Becker et al., 1990). Fluorescence in situ hybridization has been developed extensively over the last 20 years and is currently the most frequently used technique (Cavallo et al., 2007; Melo et al., 2014). Where commercial pan-centromeric FISH probes are unavailable for the species in question, eg, the rat, kinetochore labeling is required (Doherty, 2012). Fluorescence in situ hybridization has the advantage that it c (...truncated)