Upstream eventBinding, Tubulin
Altered, Chromosome number
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Directness||Weight of Evidence||Quantitative Understanding|
|Chemical binding to tubulin in oocytes leading to aneuploid offspring||indirectly leads to||Strong|
|Homo sapiens||Homo sapiens||Moderate||NCBI|
|Chinese hamsters||Cricetulus griseus||Strong||NCBI|
|Caenorhabditis elegans||Caenorhabditis elegans||Moderate||NCBI|
Life Stage Applicability
How Does This Key Event Relationship Work
In this KER chemicals that bind to tubulin indirectly lead to altered chromosome numbers. This results because of the interference of the tubulin binding leading to microtubule depolymerization, abnormal spindle structure/morphology and subsequent mis-segregation. This event can happen in vitro or in vivo, and in somatic cells or in germ cells.
Weight of Evidence
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. PARAGRAPH REVIEWING THAT PAPER ON SOMATIC PATHWAYS.
During the first meiotic division, 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]. 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].
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.
Female germ cells: 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 [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 studies we reviewed 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 Support for Linkage
Include consideration of temporal concordance here
Effects of potential aneuploidy-inducing agents on microtubule assembly in vitro has been investigated by Wallin and Hartely-Asp (1993). These authors explored the potency of XXX chemicals in binding tubulin relative to colchine, and the subsequent level of aneuploidy in these cells. The authors show - FRANC - what do they show??? Wallin and Hartley-Asp, Mutat Res 1993n 287, p17-22. FRANCEsco - insert - how much equivalent colchicine binding = how much aneuploidy. This paper should tell us at this concentration you affect microtubule assembly, then at what concentration you affect aneuploidy =also use in other indirect KER
The evidence that colchicine and other chemicals that bind to tubulin induce aneuploidy in rodent oocytes is also 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, 1987b; 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 (Table S1). 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, 1987b] , 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 of different species 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, 1987b]. 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 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 analagous 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 or Inconsistencies
We are not aware of any chemicals that bind to tubulin and do not cause aneuploidy, providing that a high enough dose/concentration was tested.
Quantitative Understanding of the Linkage
Is it known how much change in the first event is needed to impact the second? Are there known modulators of the response-response relationships? Are there models or extrapolation approaches that help describe those relationships?
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.
Evidence Supporting Taxonomic Applicability
Data for this KER are available in vitro and in vivo, and in a variety of mammalian species including humans.
Parry EM, Parry JM, Corso C, Doherty A, Haddad F, Hermine TF, Johnson G, Kayani M, Quick E, Warr T, Williamson J. Detection and characterization of mechanisms of action of aneugenic chemicals. Mutagenesis. 2002 Nov;17(6):509-21.