API

Relationship: 724

Title

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Binding, Tubulin leads to Altered, Chromosome number

Upstream event

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Binding, Tubulin

Downstream event

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Altered, Chromosome number

Key Event Relationship Overview

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AOPs Referencing Relationship

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AOP Name Adjacency Weight of Evidence Quantitative Understanding
Chemical binding to tubulin in oocytes leading to aneuploid offspring non-adjacent High

Taxonomic Applicability

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Term Scientific Term Evidence Link
Homo sapiens Homo sapiens Moderate NCBI
mouse Mus musculus High NCBI
rat Rattus norvegicus Moderate NCBI
Chinese hamsters Cricetulus griseus High NCBI
Caenorhabditis elegans Caenorhabditis elegans Moderate NCBI

Sex Applicability

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Sex Evidence
Mixed High

Life Stage Applicability

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Key Event Relationship Description

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In this KER, chemicals that bind to tubulin indirectly lead to altered chromosome numbers. This is because tubulin binding by chemicals interferes with tubulin polymerization leading to microtubule depolymerization, abnormal spindle structure/morphology and subsequent chromosome mis-segregation. The relationship is indirect because there are no studies that have measured all KEs leading up to the AO. However, as described in more details below, there are plenty of studies showing that exposure to spindle poisons induces aneuploidy in female germ cells. This relationship has been shown in vitro and in vivo, and in somatic cells as well as in germ cells.

Evidence Supporting this KER

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Strong.

Biological Plausibility

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Accurate chromosome segregation requires the temporally regulated and coordinated interaction of many cellular components including protein kinases and phosphatases, topoisomerases, the anaphase-promoting complex (APC), proteasomes, mitotic and meiotic spindle, centrosomes and kinetochores [Orr et al., 2015]. Disruption of any of these processes by chemicals can potentially result in aneuploidy [Parry et al., 2002]. There is extensive knowledge of cellular processes associated with chromosome segregation in both somatic cells [Collin et al., 2013; London et al., 2014; Musacchio, 2015] and germ cells [Polanski, 2013; Touati and Wassmann, 2016; Bennabi et al., 2016]. Although many of these cellular components and processes are shared between somatic cells and germ cells, there are features that are unique to germ cells, in general, and female germ cells specifically [Hunt and Hassold, 2002; Webster and Schuh, 2017].

Unique to germ cells are the processes that take place during the first meiotic division when homologous chromosomes must segregate to opposite poles of the cell. Homologous chromosome segregation is possible because they are paired in bivalents physically attached at chiasmata and the sister kinetochores of each chromosome are held together by complexes of cohesion proteins, behaving as a unique monooriented structure with respect to spindle microtubules [reviewed by Eichenlaub-Ritter, 2012]. This is at variance with the second meiotic division and mitotic division when segregation involves the two sister chromatids of each chromosome. Different mechanisms have been proposed to cause aneuploidy in germ cells, including: (1) nondisjunction of homologous chromosomes; (2) premature separation of homologous chromosomes or sister chromatids; and (3) recombination defects [Nagaoka et al., 2012; Zelazowski et al., 2017]. Each of these mechanisms interacts and contributes to the genesis of aneuploidy through a complex interplay of molecular and cellular events [Nagaoka et al., 2012]. Unique to female germ cells is also the formation of the meiotic spindle in the absence of centrioles, as described before, and the reduced stringency of the SAC that allows progression of meiosis even in the presence of misaligned chromosomes, and the long time that oocytes are arrested at the end of meiotic prophase with possible progressive degradation of cohesion proteins [Hunt and Hassold, 2002; Nagaoka et al., 2012; Webster and Schuh, 2017].

This KER indirectly links chemical binding to tubulin to aneuploidy. A diverse array of chemical agents are well established to induce aneuploidy, with the majority of these agents operating through binding to tubulin to impair spindle function, chromosome dynamics and ultimately segregation [reviewed in Parry et al., 2002; and in Pacchierotti and Eichenlaub-Ritter, 2011]. However, an extensive amount of work in this field has focused on gametes and since the AO of the present AOP is inherited aneuploidy, we focus on chemically-induced aneuploidy in germ cells. For a summary of chemically-induced aneuploidy in somatic cells the reader is referred to a few key reviews [e.g., Adler, 1993; Leopardi et al., 1993; Aardema et al., 1998].

There is extensive evidence in mammalian models that chemicals can induce aneuploidy by interfering with the proper functioning of the meiotic spindle and other aspects of chromosome segregation. The aneugenic activity of microtubule disrupting agents was also recently demonstrated using a Caenorhabditis elegans screening platform for the rapid assessment of chemical effects on germline function [Allard et al., 2013]. About 20 chemicals have been shown to induce aneuploidy in mammalian oocytes in vivo and the majority of these chemicals are tubulin binders (i.e., they interfere with microtubule dynamics through tubulin binding during meiosis) [Mailhes and Marchetti, 1994, 2005; Pacchierotti and Eichenlaub-Ritter, 2011]. Collectively, these studies suggest that the main window for the induction of aneuploidy in oocytes is restricted to the periovulation period with a peak of sensitivity around the resumption of meiosis and the induction of ovulation. Depending on dose and time, spindle inhibitors can induce aneuploidy in almost 100% of oocytes [reviewed in Mailhes and Marchetti, 2005], suggesting that the disruption of microtubule and spindle dynamics is a very sensitive target for the induction of aneuploidy in female germ cells. Although the majority of the available studies investigated the induction of aneuploidy during meiosis I, there is evidence that the two meiotic divisions have similar sensitivity to chemically-induced aneuploidy [Marchetti et al., 1996].

Empirical Evidence

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The effects of potential aneuploidy-inducing agents on microtubule assembly in vitro has been investigated by Brunner et al. [1991] and Wallin and Hartely-Asp [1993]. These authors explored the potency of 10 chemicals in binding tubulin relative to colchicine, and reported that there is a good correlation between the efficiency of these chemicals to interfere with microtubule assembly and their known aneugenic potential.

The evidence that colchicine and other chemicals that bind to tubulin induce aneuploidy in rodent oocytes is very strong [reviewed in Mailhes and Marchetti, 1994, 2005]. Most of the data with colchicine have been collected in mice [Tease and Fisher, 1986; Mailhes and Yuan, 1987; Mailhes et al., 1988; Mailhes et al., 1990], but evidence for colchicine-induced aneuploidy is also available in Chinese hamster [Sugawara and Mikamo, 1980] and Djungarian hamster [Hummler and Hansmann, 1985] oocytes (Annex 1). Specifically, intraperitoneal injection of 0.25 mg/kg bw colchicine before the onset of the first meiotic spindle formation increases the frequency of hyperhaploid metaphase II oocytes from 0.4 to 11.2% in mice [Tease and Fisher, 1986]. In addition, the percentage of hyperhaploid mouse oocytes induced by 0.2 mg/kg bw colchicine varies as a function of injection time [Mailhes and Yuan, 1987], with a maximum effect (10-fold increase over control level) at the time of spindle assembly (around 12 hr before ovulation). However, statistically significant effects were also reported up to 4 hr before and after this period of maximum sensitivity.

In Chinese hamsters, a single intraperitoneal injection of 3 mg/kg bw colchicine induced a 10-fold increase of hyperhaploid oocytes (from 0.8 in the control to 8.6% in the exposed group) [Sugawara and Mikamo, 1980]. Similarly, a statistically significant increase from 3.5 to 11.7% hyperhaploid oocytes was observed in Djungarian hamsters [Hummler and Hansmann, 1985]. Interestingly, the experiment with Chinese hamsters was carried out under natural ovulation conditions, i.e. without the use of exogenous hormones applied in all other studies to synchronize the oestrus cycle and increase the number of oocytes ovulated by each female. This provides experimental evidence for the lack of influence of superovulation on the aneugenic effects of colchicine. It should be noted that the same levels of meiotic arrest and aneuploidy were induced in hamsters by a dose 10 times higher than in mice. Indeed, Midgley et al. showed that hamster cells were more resistant to colchicine than mouse and human cells [Midgley et al., 1959]. While these results suggest that species-specific differences may exist in the sensitivity of oocytes to the aneugenic effects of colchicine, the consistent positive findings across species provide strong evidence to support the causal relationship between colchicine binding to tubulin and induction of aneuploidy.

Studies by Mailhes and coworkers in mice describe a dose-effect relationship for the induction of aneuploid oocytes by intraperitoneal injection of colchicine between 0.1 and 0.4 mg/kg bw [Mailhes et al., 1988; Mailhes et al., 1990], demonstrating that as the incidence of tubulin binding by colchine increases, so does the incidence of aneuploidy. Doses of 0.5 mg/kg bw and higher resulted in the arrest of all mouse oocytes at the metaphase I stage. The results for 0.2 mg/kg bw confirmed the 10-fold increase over controls reported in a previous study [Mailhes and Yuan, 1987]. At 0.3 and 0.4 mg/kg bw, the percentages of hyperhaploid oocytes increased, reaching 20.8 and 23.5%, respectively. Interestingly, no significant increase was caused by 0.1 mg/kg bw colchicine, suggesting that a threshold exists for colchicine-induced aneuploidy in mouse oocytes. The presence of a threshold for the induction of aneuploidy by tubulin binders is broadly accepted in somatic cells as well [Cammerer et al., 2010; Elhajouji et al., 2011].

The hypothesis that a certain level of tubulin damage is needed to impair spindle function is in agreement with the observation that at higher doses the “severity” of the aneugenic effect increases, with more oocytes containing not one but several supernumerary chromosomes [Mailhes et al., 1988; Mailhes et al., 1990]. Mailhes and coworkers also compared the dose-effect relationships for oocyte aneuploidy induction after intraperitoneal and oral colchicine administration [Mailhes et al., 1990]. Not surprisingly, due to the reduced bioavailability of the compound using this route, they showed that ten times higher oral doses of colchicine are needed to induce about the same level of effect induced by intraperitoneal injection. Also after oral treatment, there is a No Observed Effect Dose, corresponding, in this case, to 1 mg/kg bw. An effectiveness ratio of 10 between the two administration routes also occurs for the induction of metaphase I blocked oocytes.

Overall, about 20 chemicals have been shown to induce aneuploidy in mammalian oocytes. The majority of these chemicals are spindle poisons that are known to bind to microtubules and interact with tubulin in a manner analogous to colchicine. Data on the induction of aneuploidy in oocytes after exposure to these chemicals is reviewed in Mailhes and Marchetti [1994, 2005] and Marchetti et al. [2016].

Uncertainties and Inconsistencies

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We are not aware of any chemical that bind to tubulin and does not cause aneuploidy, providing that a high enough dose/concentration was tested.

Quantitative Understanding of the Linkage

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Although quantitative models have not been developed, the qualitative relationship is described above. In vivo studies indicate that timing prior to meiotic division must be carefully considered because of the different half lives of the chemicals bound to tubulin. Depending on dose and time, chemicals that bind to tubulin can induce aneuploidy in almost 100% of oocytes [Mailhes and Marchetti, 2005], suggesting that the disruption of microtubule and spindle dynamics is a very sensitive target for the induction of aneuploidy in female germ cells.

Response-response Relationship

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Time-scale

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Known modulating factors

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Known Feedforward/Feedback loops influencing this KER

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Domain of Applicability

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Data for this KER are available in vitro and in vivo, and in a variety of mammalian species including humans.

References

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Adler ID. 1993. Synopsis of the in vivo results obtained with the 10 known or suspected aneugens tested in teh CEC collaborative study. Mutat Res 287:131-137.

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.

Bennabi I, Terret ME, Verlhac MH. 2016. Meiotic spindle assembly and chromosome segregation in oocytes. J Cell Biol 215:611-619.

Brunner M, Albertini S, Würgler FE. 1991. Effects of 10 known or suspected spindle poisons in the in vitro porcine brain tubulin assembly assay. Mutagen 6:65-70.

Cammerer Z, Schumacher MM, Kirsch-Volders M, Suter W, Elhajouji A. 2010. Flow cytometry peripheral blood micronucleus test in vivo: determination of potential threshold for aneuploidy induced by spindle poisons. Environ Mol Mutagen 51:278-284.

Collin P, Nashchekina O, Walker R, Pines J. 2013. The spindle assembly checkpoint works like a rheostat rather than a toggle switch. Nat Cell Biol 15:1378–1385.

Eichenlaub-Ritter U. 2012. Female meiosis and beyond: more questions than answers? Reprod Biomed Online 24:589-590.

Elhajouji A, Lukamowicz M, Cammerer Z, Kirsch-Volders M. 2011. Potential thresholds for genotoxic effects by micronucleus scoring. Mutagenesis 26:199-204.

Hassold T, Hall H, Hunt P. 2007. The origin of human aneuploidy: Where we have been, where we are going. Hum Mol Genet 16: R203–R208.

Hummler E, Hansmann I. 1985. Preferential nondisjunction of specific bivalents in oocytes from Djungarian hamsters (Phodopus sungorus) following colchicine treatment. Cytogenet Cell Genet 39:161–167.

Hunt PA, Hassold TJ. 2002. Sex matters in meiosis. Science 296:2181–2183.

Leopardi P, Zijno A, Bassani B, Pacchierotti F. 1993. In vivo studies on chemically induced aneuploidy in mouse somatic and germinal cells. Mutat Res 287:119-130.

London N, Biggins S. 2014. Signalling dynamics in the spindle checkpoint response. Nat Rev Mol Cell Biol 15:736–747.

Mailhes JB, Marchetti F. 1994. The influence of postovulatory ageing on the retardation of mouse oocyte maturation and chromosome segregation induced by vinblastine. Mutagenesis 9:541–545.

Mailhes JB, Marchetti F. 2005. Mechanisms and chemical induction of aneuploidy in rodent germ cells. Cytogenet Genome Res 111: 384–391.

Mailhes JB, Yuan ZP. 1987. Differential sensitivity of mouse oocytes to colchicine-induced aneuploidy. Environ Mol Mutagen 10:183–188.

Mailhes JB, Preston RJ, Yuan ZP, Payne HS. 1988. Analysis of mouse metaphase II oocytes as an assay for chemically induced aneuploidy. Mutat Res 198:145–152.

Mailhes JB, Yuan ZP, Aardema MJ. 1990. Cytogenetic analysis of mouse oocytes and one-cell zygotes as a potential assay for heritable germ cell aneuploidy. Mutat Res 242:89–100.

Marchetti F, Mailhes JB, Bairnsfather L, Nandy I, London SN. 1996. Dose-response study and threshold estimation of griseofulvininduced aneuploidy during female mouse meiosis I and II. Mutagenesis 11:195–200.

Midgley AR, Pierce B, Dixon FJ. 1959. Nature of colchicine resistance in golden hamster. Science 130:40–41.

Musacchio A. 2015. The molecular biology of spindle assembly checkpoint signaling dynamics. Curr Biol 25:R1002-R1018.

Nagaoka SI, Hodges CA, Albertini DF, Hunt PA. 2011. Oocyte-specific differences in cell-cycle control create an innate susceptibility to meiotic errors. Curr Biol  21:651-657.

Nagaoka SI, Hassold TJ, Hunt PA. 2012. Human aneuploidy: Mechanisms and new insights into an age-old problem. Nat Rev Genet 13:493–504.

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Pacchierotti F, Eichenlaub-Ritter U. 2011. Environmental hazard in the aetiology of somatic and germ cell aneuploidy. Cytogenet Genome Res 133:254-268.

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Tease C, Fisher G. 1986. Oocytes from young and old female mice respond differently to colchicine. Mutat Res 173:31–34.

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