Key Event Title
|Level of Biological Organization|
|female germ cell|
Key Event Components
|abnormal chromosome number||increased|
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|Tubulin binding and aneuploidy||KeyEvent|
Key Event Description
This key event describes the presence of an abnormal number of chromosomes in cells (i.e., aneuploidy) that is different from the haploid number or its multiples.
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 and there are OECD test guidelines for cytogenetic analysis of chromosome abnormalities in somatic cells both in vitro [OECD, 2016a] and in vivo [OECD, 2016b]. Although, the detection of aneuploidy for regulatory purposes is not standardized using these approaches, these methods are routinely used in research studies [Aardema et al, 1998]. Aneugens can be detected using the micronucleus assay [OECD, 2016c,d], but these methods are not specific to aneugens. Integration of centromere-specific probes in micronucleus assays enables assessment of aneugenicity using these approaches [Zijno et al., 1996]. Recently, flow cytometry approaches have been developed that are using multiple endpoints to discriminate aneugens from other classes of chemicals [Bryce et al., 2014].
Methods for handling either single oocytes [Tarkowski, 1966] or multiple oocytes [Mailhes and Yuan, 1987] 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], fluorescent DNA immunostaining [Leland et al., 2009] or spectral karyotyping [Márquez et al., 1998]. 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].
The cytogenetic analysis of oocytes can also identify the presence of single chromatids originated because of premature sister chromatid separation (PSCS), which is one of the main mechanisms thought to result in aneuploidy in human oocytes [Angell, 1997]. This is has now been demonstrated in mouse oocytes as well [Yun et al., 2014]. The presence of single chromatids in an otherwise normal oocyte can predispose to the induction of aneuploidy during the second meiotic division. In fact, there is one example of a chemical treatment that did not increase aneuploidy in oocytes, but it did so in zygotes because of the presence of PSCS in oocytes [Mailhes et al., 1997].
Oocytes of several rodent species [reviewed in Mailhes and Marchetti, 2005; Pacchierotti et al., 2007] and human oocytes [Pellestor et al., 2005] have been analyzed for assessing aneuploidy. 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.
Domain of Applicability
Aneuploidy has been measured in many cells types of mammals [Aardema et al., 1998; Mailhes and Marchetti, 1994; 2005; Marchetti et al., 2016], model organisms [Allard et al., 2013; Birchler, 2013] and unicellular organisms [Strome and Plon, 2010]. Therefore, this key event is relevant to all eukaryotic organisms.
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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.
Angell R. 1997. First-meiotic-division non-disjunction in human oocytes. Am J Hum Genet 61:23-32.
Birchler JA. 2013. Aneuploidy in plants and flies: the origin of studies of genomic imbalance. Semin Cell Dev Biol 24:315-319.
Bryce SM, Bemis JC, Mereness JA, Spellman RA, Moss J, Dickinson D, Schuler MJ, Dertinger SD. 2014. Interpreting in vitro micronucleus positive results: simple biomarker matrix discriminates clastogens, aneugens, and misleading positive agents. Environ Mol Mutagen 55:542-555.
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. 1987. Cytogenetic technique for mouse metaphase II oocytes. Gamete Res 18:77-83.
Mailhes JB, Marchetti F. 1994. Chemically-induced aneuploidy in mammalian oocytes. Mutat Res 320:87-111.
Mailhes JB, Marchetti F. 2005. Mechanisms and chemically-induced aneuploidy in rodent germ cells. Cytogenet Genome Research 111:384-391.
Mailhe JB, Young D, London SN. 1997. 1,2-Propanediol-induced premature centromere separation in mouse oocytes and aneuploidy in one-cell zygotes. Biol Reprod 57:92-98.
Marchetti F, Massarotti A, Yauk CL, Pacchierotti F, Russo A. 2016. The adverse outcome pathway (AOP) for chemical binding to tubulin in oocytes leading to aneuploid offspring. Environ Mol Mutagen 57:87-113.
Márquez C, Cohen J, Munné S. 1998. Chromosome identification in human oocytes and polar bodies by spectral karyotyping. Cytogenet Cell Genet 81:254-258.
Mulla W, Zhu J, Li R. 2014. Yeast: a simple model system to study complex phenomena of aneuploidy. FEMS Microbiol Rev 38:201-212.
OECD. 2016a. Test No. 473: In Vitro Mammalian Chromosomal Aberration Test, OECD Publishing, Paris. http://dx.doi.org/10.1787/9789264264649-en.
OECD. 2016b. Test No. 475: Mammalian Bone Marrow Chromosomal Aberration Test, OECD Publishing, Paris. http://dx.doi.org/10.1787/9789264264786-en.
OECD. 2016c. Test No. 474: Mammalian Erythrocyte Micronucleus Test, OECD Publishing, Paris. http://dx.doi.org/10.1787/9789264264762-en.
OECD. 2016d. Test No. 487: In Vitro Mammalian Cell Micronucleus Test, OECD Publishing, Paris. http://dx.doi.org/10.1787/9789264264861-en.
Pacchierotti F, Adler ID, Eichenlaub-Ritter U, Mailhes JB. 2007. Gender effects on the incidence of aneuploidy in mammalian germ cells. Environ Res 104:46-69.
Pellestor F, Anahory T, Hamamah S. 2005. The chromosomal analysis of human oocytes. An overview of established procedures. Human Reprod Update 11:15-32.
Salamanca F, Armendares S. 1974. C bands in human metaphase chromosomes treated by barium hydroxide. Ann Genet 17:135-136.
Strome ED, Plon SE. 2010. Utilizing Saccharomyces cerevisiae to identify aneuploidy and cancer susceptibility genes. Methods Mol Biol 653:73-85.
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.
Yun Y, Lane SI, Jones KT. 2014. Premature dyad separation in meiosis II is the major segregation error with maternal age in mouse oocytes. Development 141:199-208.
Zijno A, Marcon F, Leopardi P, Crebelli R. 1996. 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