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Binding, Tubulin leads to Altered, Chromosome number
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding||Point of Contact||Author Status||OECD Status|
|Chemical binding to tubulin in oocytes leading to aneuploid offspring||non-adjacent||High||Francesco Marchetti (send email)||Open for citation & comment||EAGMST Under Review|
Life Stage Applicability
|All life stages||Moderate|
Key Event Relationship Description
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 Collection Strategy
Evidence Supporting this KER
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 thus, 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].
The effects of potential aneuploidy-inducing agents on microtubule assembly in vitro has been investigated by Brunner et al.  and Wallin and Hartely-Asp . 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 (Supplementary Table 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. .
Uncertainties and Inconsistencies
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.
Known modulating factors
As described above, time of exposure with respect to ovulation is a modulating factor.
Quantitative Understanding of the Linkage
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.
It is difficult to compare the response-response relationship between these two KEs, as binding to tubuline (KEupstream) is generally measured in an acellular system or in vitro, while altered chromosome nubmer (KEdownstream) is measured in vivo. However, Brunner et al.  and Wallin and Hartely-Asp  analyzing the ability of 10 chemicals to interfere with microtubule assembly reported that there is a good correlation between the efficiency of microtubule assembly interference and the anuegenic potential of each chemical. That is, chemicals that interfered with microtubule assembly at low concentrations are strong aneugens (eg, colchicine, vinblastine); while chemicals that did not affect the steady state of microtubule assembly do not induced aneuploidy or are very weak inducers (eg, diazepam, cadmium chloride).
Binding to tubulin is occurring on the time scale seconds (acellular systems) and minutes (in vitro). In vivo, the time-scale is determined by the route of administration and the ADME characteristics of the chemical. For the induction of aneuploidy, chemical binding to tubulin must occur within a short time range before the completion of the first meiotic division. Mailhes and Yuan  showed that the induction of aneuploid oocytes following exposure to colchicine is maximum when administered 12 hr before ovulation and is reduced when given more or less than 12 hours.
Known Feedforward/Feedback loops influencing this KER
No known feedback loops.
Domain of Applicability
Data for this KER are available in vitro and in vivo, and in a variety of mammalian species including humans.
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