Key Event Overview
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AOPs Including This Key Event
|AOP Name||Event Type||Essentiality|
|Chemical binding to tubulin in oocytes leading to aneuploid offspring||KE|
Level of Biological Organization
How this Key Event works
This key event describes the presence of an abnormal number of chromosomes in cells (i.e., aneuploidy).
How it is Measured or Detected
Aneuploidy (i.e., altered chromosome number) is assessed by standard cytogenetic methods that entail the preparation of meiotic or mitotic metaphases to count the number of chromosomes present. Standard methods for assessment in somatic cells have been described [INSERT REFERENCES]. There are OECD guidelines for cytogenetic analysis of chromosome abnormalities in somatic cells [OECD TG 473 and 475); however, the detection of aneuploidy is not standardized using these approaches. Aneugens are detected by the micronucleus assay [OECD TG 474 and 487], but these methods are not specific to only aneugens. Integration of centromere-specific probes in micronucleus assays enables assessment of aneugenicity using these approaches (Zijno et al. 1996].
Methods for handling either single oocytes [Tarkowski 1966] or multiple oocytes [Mailhes and Yuan 1987a] are available. Metaphases are then analyzed under a microscope to count the number of chromosomes. To improve the accuracy of counting, identification of the centromeres can be done using traditional C-banding [Salamanca and Armendares 1974] or fluorescent immunostaining [Leland et al. 2009]. In these studies, the analyzed endpoint is the chromosome number in either second meiotic metaphases or zygotic metaphases. According to a conservative approach, evidence of aneuploidy induction is provided by a statistically significant increase of hyperhaploid metaphases because it cannot be excluded that some hypohaploid metaphases may result from technical artifacts. However, chromosome nondisjunction is expected to produce equal numbers of hyper- or hypohaploid oocytes. Thus, to estimate the total frequency of aneuploid oocytes induced by this mechanism, the frequency of hyperhaploid metaphases is generally doubled. Even this calculation may lead to an underestimate of the absolute aneugenic effect because mechanisms other than nondisjunction, such as chromosome lagging, may produce an excess of hypohaploidies. Indeed, an excess of colchicine-induced hypohaploid oocytes has been reported [Sugawara and Mikamo 1980].
Aneugenicity can also be measured using a C. elegans screening platform for rapid assessment [Allard et al. 2013]. This methodology fluorescently marks aneuploid eggs and embryos.
Consider the following criteria when describing each method: 1. Is the assay fit for purpose? Yes 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? Directly 3. Is the assay repeatable? Yes 4. Is the assay reproducible? Yes
Evidence Supporting Taxonomic Applicability
This key event is relevant to all eukaryotic organisms.
Allard P, Kleinstreuer NC, Knudsen TB, Colaiacovo MP. 2013. A C. elegans screening platform for the rapid assessment of chemical disruption of germline function. Environ Health Perspect 121:717-724.
Leland S, Nagarajan P, Polyzos A, Thomas S, Samaan G, Donnell R, Marchetti F, Venkatachalam S. 2009. Heterozygosity for a Bub1 mutation causes female-specific germ cell aneuploidy in mice. Proc Natl Acad Sci USA 106:12776-12781.
Mailhes JB, Yuan ZP. 1987a. Cytogenetic technique for mouse metaphase II oocytes. Gamete Res 18:77-83.
OECD (1997), Test No. 473: In vitro Mammalian Chromosome Aberration Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris. DOI: http://dx.doi.org/10.1787/9789264071261-en
OECD (1997), Test No. 474: Mammalian Erythrocyte Micronucleus Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris. DOI: http://dx.doi.org/10.1787/9789264071285-en
OECD (2014), Test No. 475: Mammalian Bone Marrow Chromosomal Aberration Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris. DOI: http://dx.doi.org/10.1787/9789264224407-en
OECD (2010), Test No. 487: In Vitro Mammalian Cell Micronucleus Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris. DOI: http://dx.doi.org/10.1787/9789264091016-en
Salamanca F, Armendares S. 1974. C bands in human metaphase chromosomes treated by barium hydroxide. Ann Genet 17:135-136.
Sugawara S, Mikamo K. 1980. An experimental approach to the analysis of mechanisms of meiotic nondisjunction and anaphase lagging in primary oocytes. Cytogenet Cell Genet 28:251-264.
Tarkowski AK. 1966. An Air-Drying Method for Chromosome Preparations from Mouse Eggs. Cytogenetic and Genome Research 5:394-400.
Zijno A, Marcon F, Leopardi P, Crebelli R. Analysis of chromosome segregation in cytokinesis-blocked human lymphocytes: non-disjunction is the prevalent damage resulting from low dose exposure to spindle poisons. Mutagenesis. 1996 Jul;11(4):335-40.