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Event: 723

Key Event Title

The KE title should describe a discrete biological change that can be measured. It should generally define the biological object or process being measured and whether it is increased, decreased, or otherwise definably altered relative to a control state. For example “enzyme activity, decreased”, “hormone concentration, increased”, or “growth rate, decreased”, where the specific enzyme or hormone being measured is defined. More help

Altered, Chromosome number

Short name
The KE short name should be a reasonable abbreviation of the KE title and is used in labelling this object throughout the AOP-Wiki. The short name should be less than 80 characters in length. More help
Altered, Chromosome number

Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. Note, KEs should be defined within a particular level of biological organization. Only KERs should be used to transition from one level of organization to another. Selection of the level of biological organization defines which structured terms will be available to select when defining the Event Components (below). More help
Level of Biological Organization
Cellular

Cell term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help
Cell term
female germ cell

Organ term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help

Key Event Components

Further information on Event Components and Biological Context may be viewed on the attached pdf.Because one of the aims of the AOP-KB is to facilitate de facto construction of AOP networks through the use of shared KE and KER elements, authors are also asked to define their KEs using a set of structured ontology terms (Event Components). In the absence of structured terms, the same KE can readily be defined using a number of synonymous titles (read by a computer as character strings). In order to make these synonymous KEs more machine-readable, KEs should also be defined by one or more “event components” consisting of a biological process, object, and action with each term originating from one of 22 biological ontologies (Ives, et al., 2017; See List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling). The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signalling by that receptor).Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description. To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons. If a desired term does not exist, a new term request may be made via Term Requests. Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add. More help
Process Object Action
abnormal chromosome number increased

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE. Clicking on the name of the AOP will bring you to the individual page for that AOP. More help
AOP Name Role of event in AOP Point of Contact Author Status OECD Status
Tubulin binding and aneuploidy KeyEvent Cataia Ives (send email) Open for citation & comment EAGMST Under Review

Stressors

This is a structured field used to identify specific agents (generally chemicals) that can trigger the KE. Stressors identified in this field will be linked to the KE in a machine-readable manner, such that, for example, a stressor search would identify this as an event the stressor can trigger. NOTE: intermediate or downstream KEs in one AOP may function as MIEs in other AOPs, meaning that stressor information may be added to the KE description, even if it is a downstream KE in the pathway currently under development.Information concerning the stressors that may trigger an MIE can be defined using a combination of structured and unstructured (free-text) fields. For example, structured fields may be used to indicate specific chemicals for which there is evidence of an interaction relevant to this MIE. By linking the KE description to a structured chemical name, it will be increasingly possible to link the MIE to other sources of chemical data and information, enhancing searchability and inter-operability among different data-sources and knowledgebases. The free-text section “Evidence for perturbation of this MIE by stressor” can be used both to identify the supporting evidence for specific stressors triggering the MIE as well as to define broad chemical categories or other properties that classify the stressors able to trigger the MIE for which specific structured terms may not exist. More help

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected from an ontology. In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
Term Scientific Term Evidence Link
mouse Mus musculus High NCBI
Hamster Hamster Moderate NCBI
Homo sapiens Homo sapiens Low NCBI

Life Stages

The structured ontology terms for life-stage are more comprehensive than those for taxa, but may still require further description/development and explanation in the free text section. More help
Life stage Evidence
All life stages Moderate

Sex Applicability

The authors must select from one of the following: Male, female, mixed, asexual, third gender, hermaphrodite, or unspecific. More help
Term Evidence
Mixed Moderate

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. For example, the biological state being measured could be the activity of an enzyme, the expression of a gene or abundance of an mRNA transcript, the concentration of a hormone or protein, neuronal activity, heart rate, etc. The biological compartment may be a particular cell type, tissue, organ, fluid (e.g., plasma, cerebrospinal fluid), etc. The role in the biology could describe the reaction that an enzyme catalyses and the role of that reaction within a given metabolic pathway; the protein that a gene or mRNA transcript codes for and the function of that protein; the function of a hormone in a given target tissue, physiological function of an organ, etc. Careful attention should be taken to avoid reference to other KEs, KERs or AOPs. Only describe this KE as a single isolated measurable event/state. This will ensure that the KE is modular and can be used by other AOPs, thereby facilitating construction of AOP networks. More help

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

One of the primary considerations in evaluating AOPs is the relevance and reliability of the methods with which the KEs can be measured. The aim of this section of the KE description is not to provide detailed protocols, but rather to capture, in a sentence or two, per method, the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements. Methods that can be used to detect or measure the biological state represented in the KE should be briefly described and/or cited. These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA).Key considerations regarding scientific confidence in the measurement approach include whether the assay is fit for purpose, whether it provides a direct or indirect measure of the biological state in question, whether it is repeatable and reproducible, and the extent to which it is accepted in the scientific and/or regulatory community. Information can be obtained from the OECD Test Guidelines website and the EURL ECVAM Database Service on Alternative Methods to Animal Experimentation (DB-ALM). ?

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

This free text section should be used to elaborate on the scientific basis for the indicated domains of applicability and the WoE calls (if provided). While structured terms may be selected to define the taxonomic, life stage and sex applicability (see structured applicability terms, above) of the KE, the structured terms may not adequately reflect or capture the overall biological applicability domain (particularly with regard to taxa). Likewise, the structured terms do not provide an explanation or rationale for the selection. The free-text section on evidence for taxonomic, life stage, and sex applicability can be used to elaborate on why the specific structured terms were selected, and provide supporting references and background information.  More help

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.

Evidence for Perturbation by Stressor

Colchicine

Ten fold significant increase of hyperhaploid oocytes. 8.6% (30/342) hyperhaploid oocytes vs 0.8% (14/1730) in controls. Oocytes collected aftern natural ovulation, strenghtening the relevance of data for human hazard  assessment (Sugawara and Mikamo, 1980)

In Djungarian hamsters, 3 mg/kg Colchicine 5 hours after induction of ovulation induces a significant increase of hyperhaploid oocytes. 11.7% (16/137) hyperhaploid oocytes vs 3.5 in controls (Hummler and Hansmann, 1985).

In mice, 0.25 mg/kg Colchicine significantly increased hyperhaploid oocytes in both young and old female (Tease and Fisher, 1986). In another study, 0.2 mg/kg colchicine at diffrent times from the induction of ovulation (-4 hr to +4 hr) significantly increased hyperhaploid oocyte at all timepoint invegated (Mailhes and Yuan, 1987). This study shows that in preovulatory oocytes the sensitivity window for the induction of aneuploidy is at least 8 hr long. In a subsequent study, a dose-related increase in hyperhaploid oocytes was found (Maihes et al 1988). FInally, another study demonstrated that an aneuploidy induction effectiveness ratio of 10 is observed between administering colchicine orally or by intraperiotoneal injection (Mailhes et al 1990)

2-Methoxyestradiol

Dose related increases in hyperhaploid oocytes after in vitro treatement. The lowest effective tested concentration was 3.75 microM (Eichenlaub-Ritter et al 2007). This study provides evidence that spindle and chromosome congression defects precede the observation of aneuploid oocytes.

Podophyllotoxin

Administration of 20 mg/kg podophillotoxin at the onset of the first meiotic spindle formation (ie 16 hours before oocyte collection, induced a statistically significant increase in hyperhaploid oocytes from chinese hamsters (Tateno et al 1985)

Nocodazole

In vitro expsoure to nocodazole for one hour during the first meiotic spindel formation induces a statistically significant increase in hyperploid mouse oocytes (Eichenlaub-Ritter and Boll, 1989). Subsequently, a dose-dependent increase in hyperhaploidy oocytes was found (Shen et al 2005); The lowest effective concentration for aneuploidy induction in metaphase II is 40 nM. This paper provides evidence of aneuploid linked to evidence of spindle  and chromosome congression defects with a dose response relationship. The study of Sun et al (2005) confirmed the dose-dependent increase in hyperhaploid oocytes and showed that oocytes enclosed in their follicle appear more sensitive than denude oocytes to the aneugenic activity of nocodazole

In vivo, administration of 70 mg/kg nocodazole at the time of the induction of ovulation significantly increased hyperhaploid oocytes while a dose of 35 mg/kg did not (Sun et al 2005)

Benomyl

Administration of benomyl ranging from 500 to  2000 mg/kg per os at the time of the induction of ovulation increased hyperhaploidy mouse oocytes at all doses tested (Mailhes and Aardema, 1992). A saturation of the effect is detected for doses above 1500 mg/kg

Carbendazim

A dose of 1000 mg/kg carbendazin administered per os either 4.5 or 6 hr after induction of ovulation significantly increased hyperhaploidy oocytes in Djungarian hamster (Hummler and Hansmann, 1988). The same dose administered at the time of ovulation induction induced a 4-fold increase in hyperhaploidy oocytes over the control values in Syrian Hamsters (JEffay et al 1996).

Thiabendazole

Thiabendazole was tested in mice at doses ranging 50 to 150 mg/kg. Small but significant increase in hyperhaploid oocytes was found at 100 mg/kg (Mailhes et al 1997)

Vinblastine sulfate

Vinblastine was tested in mice  at doses ranging from 0.9 to 9 mg/kg. Significant increses in hyperhaploid oocytes were seen at 0.23 and 0.45 mg/kg (Russo and Pacchierotti, 1988). Higher doses arrested all oocytes at the metaphase I stage, thus, preventing the manifestation of aneuploidy. These results were confirmed in another study (Maihles et al 1993). A study that followed the fate of arrested oocytes, showed that delaying collection of oocytes resulted in a reduction in metaphase I oocytes and a corresponding increase in diploid oocytes (Maihles and Marchetti, 1994).

A study in chinese hamster (Tateno et al 1995) showed that the increase in hyperhaploidy oocytes is similar to what is observed in the mouse with a dose that is 10 times lower.

References

List of the literature that was cited for this KE description. Ideally, the list of references, should conform, to the extent possible, with the OECD Style Guide (https://www.oecd.org/about/publishing/OECD-Style-Guide-Third-Edition.pdf) (OECD, 2015). More help

Aardema MJ, Albertini S, Arni P, Henderson LM, Kirsch-Volders M, Mackay JM, Sarrif AM, Stringer DA, Taalman RD. 1998. Aneuploidy: a report of an ECETOC task force. Mutat Res 410:3-79.

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