Francesco Marchetti 1*, Alberto Massarotti 2, Carole L. Yauk 1, Francesca Pacchierotti 3, Antonella Russo 4
1 Environmental Health Science and Research Bureau, Health Canada, Ottawa, Canada. 2 Dipartimento di Scienze del Farmaco, Università degli Studi del Piemonte Orientale “A. Avogadro”, Novara, Italy. 3 Unit of Radiation Biology and Human Health, Laboratory of Toxicology, ENEA CR Casaccia, Rome, Italy. 4 Department of Biology, University of Padova, Padova, Italy.
- Correspondence to: Francesco Marchetti, Environmental Health Science and Research Bureau, Health Canada, Ottawa, ON K1A 0K9, Canada. E-mail: firstname.lastname@example.org
Point of Contact
- Francesco Marchetti
|Author status||OECD status||OECD project||SAAOP status|
|Under development: Not open for comment. Do not cite||Under Development|
This AOP was last modified on December 12, 2016 15:25
|Binding, Tubulin||September 16, 2017 10:16|
|Depolymerization, Microtubule||September 16, 2017 10:16|
|Disorganization, Spindle||September 16, 2017 10:16|
|Increase, Aneuploid offspring||November 29, 2016 19:22|
|Altered, Meiotic chromosome dynamics||September 16, 2017 10:16|
|Altered, Chromosome number||September 16, 2017 10:16|
|Binding, Tubulin leads to Depolymerization, Microtubule||December 12, 2016 10:56|
|Depolymerization, Microtubule leads to Disorganization, Spindle||December 12, 2016 11:00|
|Altered, Chromosome number leads to Increase, Aneuploid offspring||December 12, 2016 11:07|
|Binding, Tubulin leads to Altered, Chromosome number||December 12, 2016 11:25|
|Disorganization, Spindle leads to Altered, Meiotic chromosome dynamics||December 12, 2016 13:44|
|Altered, Meiotic chromosome dynamics leads to Altered, Chromosome number||December 12, 2016 14:42|
|Colchicine||November 29, 2016 18:42|
Aneuploidy, an abnormal number of chromosomes, arising during meiosis in germ cells represents the most common chromosomal abnormality at birth and is the leading cause of pregnancy loss in humans. Aneuploidy can affect any chromosome, and data in rodents suggest that neither aneuploid sperm nor aneuploid oocytes are selected against at fertilization. Therefore, an increase in germ cell aneuploidy is expected to result in an increase in aneuploid pregnancies. The etiology of human aneuploidy is still not well understood, although there is strong evidence supporting a preferential occurrence during female meiosis I and a positive correlation with maternal age. There is extensive evidence in animal models that chemicals can induce aneuploidy by interfering with the proper functioning of the meiotic spindle and other aspects of chromosome segregation. Over 15 chemicals have been shown to induce aneuploidy in mammalian oocytes and the majority of these chemicals interfere with microtubule dynamics during meiosis. In addition to these animals studies, there is also one reported case in which environmental exposure to trichlorfon, an organophosphate insecticide, resulted in a cluster of Down syndrome cases among women in an Hungarian community. The present AOP focuses on the induction of aneuploidy in mammalian oocytes as a consequence of chemical binding to tubulin (MIE). In this AOP, chemicals that bind to tubulin lead to the depolymerization of microtubules (KE1). Extensive microtubule depolymerisation leads to meiotic spindle disorganization (KE2), which in turns lead to altered chromosome dynamics (KE3) and the generation of aneuploidy oocytes (KE4). Aneuploidy oocytes can be fertilized and generate aneuploidy offspring (AO). There is ample empirical evidence supporting this AOP and the overall weight of evidence is strong.
Aneuploidy is associated with serious human health effects. Approximately 10–30% of human zygotes, 50% of spontaneous abortions, and 0.3% of human newborns are aneuploid [Hassold et al. 2007; Nagaoka et al. 2012]. Cytogenetic analyses of human oocytes and preimplantation embryos have reported frequencies of aneuploidy in excess of 50% [Magli et al. 2001; Munne 2002; Kuliev et al. 2003]. In these studies, the overall aneuploidy frequency is estimated from the analysis of a subset of chromosomes, which may affect the accuracy of the estimate.
Aneuploidy can affect any chromosome [Nagaoka et al. 2012], although there is evidence that acrocentric chromosomes may be more frequently involved in aneuploidy than metacentric chromosomes [Nicolaidis and Petersen 1998; Hassold et al. 2007; Gianaroli et al. 2010]. In humans, only trisomies for a few autosomal chromosomes (13, 18 and 21) and aneuploidies of the sex chromosomes are compatible with life. These aneuploidies have important developmental, neurological and reproductive effects. Trisomy 21 or Down syndrome, with an occurrence of ~1/720 births, is the most common genetic abnormality in newborns [Hassold et al. 2007].
Summary of the AOP
Molecular Initiating Event
|Binding, Tubulin||Binding, Tubulin|
|Depolymerization, Microtubule||Depolymerization, Microtubule|
|Disorganization, Spindle||Disorganization, Spindle|
|Altered, Meiotic chromosome dynamics||Altered, Meiotic chromosome dynamics|
|Altered, Chromosome number||Altered, Chromosome number|
|Increase, Aneuploid offspring||Increase, Aneuploid offspring|
Relationships Between Two Key Events (Including MIEs and AOs)
|Binding, Tubulin leads to Depolymerization, Microtubule||Directly leads to||Strong|
|Depolymerization, Microtubule leads to Disorganization, Spindle||Directly leads to||Moderate|
|Altered, Chromosome number leads to Increase, Aneuploid offspring||Directly leads to||Strong|
|Binding, Tubulin leads to Altered, Chromosome number||Indirectly leads to||Strong|
|Disorganization, Spindle leads to Altered, Meiotic chromosome dynamics||Directly leads to||Moderate|
|Altered, Meiotic chromosome dynamics leads to Altered, Chromosome number||Directly leads to||Weak|
Life Stage Applicability
|Adult, reproductively mature||Strong|
|Mus musculus||Mus musculus||Strong||NCBI|
|Homo sapiens||Homo sapiens||Moderate||NCBI|
Graphical RepresentationClick to download graphical representation template
Overall Assessment of the AOP
Domain of Applicability
The present AOP should be considered specific to female germ cells exposed in the peri-ovulation period. The majority of data on this AOP were derived from experiments in mice. The available results on aneuploidy induction induced by the prototype tubulin-binding chemical colchicine in oocytes of species other than Mus musculus are qualitatively consistent with mouse data, in agreement with the similarities in the mechanism of action across several Phyla and the high degree of homology of tubulin across species. Evidence for microtubule depolymerization and spindle disorganization has been obtained in human oocytes exposed in culture to colchicine. This suggests that the MIE and KEs are conserved and would occur in human oocytes also. Therefore, the AOP should apply to any species that produce eggs.
Essentiality of the Key Events
Not all events within this AOP can be tested for essentiality. This is due to technical limitations at this time. However, there is one study demonstrating the essentiality of proper spindle organization for correct chromosome congression and segregation. Ou et al.  showed that depletion of the microtubule organizing centres (required for spindle organization) leads to increase in the incidence spindle and chromosome dynamic abnormalities. Moreover, studies with mice deficient in specific spindle assembly checkpoint proteins have an increase in the occurrence of high levels of aneuploid oocytes in these mice [McGuinness et al., 2009; reviewed in Mailhes and Marchetti, 2010].
Weight of Evidence Summary
Biological plausibility: The extensive knowledge about mechanisms of chromosome segregation in meiosis, and the experimental data on the effects of tubulin binders on aneuploidy induction in rodent oocytes provide overall sound strong biological plausibility to this AOP. The relationship between altered chromosomal alignment and segregation (KE3) and the generation of aneuploid oocytes (KE4) is the weakest link of the AOP.
Empirical support: Overall, the timescale of events, from the initial biochemical interactions (MIE) occurring within seconds to minutes of exposure, through disruption of spindle (KE2) and chromosome alignment and segregation in meiosis (KE3) occurring in the following hours, to the formation and ovulation of an aneuploid oocyte (KE4) and to its possible fertilization, which would occur later on, is fully coherent and consistent with the timeline of oocyte development and fertilization [Marchetti et al., 2016]. Moreover, examination of the incidence of events occurring across doses for KE2, KE3 and KE4 after in vitro exposure of oocytes to nocodazole [Shen et al., 2005] and 2-methoxyestradiol [Eichenlaub-Ritter et al., 2007] supports the order and linkages between the KEs across the AOP.
The comparison between the lowest effective concentrations inducing each subsequent event is complex because colchicine binding to tubulin and microtubule depolymerization are measured in acellular systems, whereas, spindle disorganization and altered chromosome alignment and segregation are mostly analysed in cultured oocytes, and induction of aneuploid oocytes and zygotes is assessed after treatment of laboratory rodents by intraperitoneal or oral administrations. Cells in culture may respond to chemical exposure with a different sensitivity than whole organisms [Sun et al., 2005], and a comparison between in vitro molar concentrations and mg/kg body weight of in vivo administered doses can be done only roughly, based on many assumptions. Furthermore, few in vitro experiments were aimed at identifying the Lowest Effective Tested Concentration, or were even conducted at multiple concentration levels. In many cases, experiments aimed to test the hypothesis that a given effect was elicited by chemical disruption of a certain process, and to do this, high doses were used. The published work shows that there is progressivity between dose, severity of spindle damage and degree of aneuploidy, from one to several involved chromosomes up to a complete inhibition of chromosome segregation and arrest of oocytes at meiosis I [Russo and Pacchierotti, 1988; Mailhes et al., 1990; Mailhes and Aardema, 1992; Mailhes et al., 1993a; Sun et al., 2005; Eichenlaub-Ritter et al., 2007].
Quantitative ConsiderationsThe extent of quantitative understanding of the various KERs in the overall hypothesised AOP is also critical in consideration of potential regulatory application. For some applications (e.g. doseresponse analysis in in depth risk assessment), quantitative characterisation of downstream KERs may be essential while for others, quantitative understanding of upstream KERs may be important (e.g., QSAR modelling for category formation for testing). Because evidence that contributes to quantitative understanding of the KER is generally not mutually exclusive with the empirical support for the KER, evidence that contributes to quantitative understanding should generally be considered as part of the evaluation of the weight of evidence supporting the KER (see Annex 1, footnote b). General guidance on the degree of quantitative understanding that would be characterised as weak, moderate, or strong is provided in Annex 2. Instructions To edit the “Quantitative Considerations” section, on an AOP page, in the upper right hand menu, click ‘Edit.’ This brings you to a page entitled, “Editing AOP.” Scroll down to the “Quantitative Considerations” section, where a text entry box allows you to submit text. In the upper right hand menu, click ‘Update AOP’ to save your changes and return to the AOP page. The new text should appear under the “Quantitative Considerations” section on the AOP page.
Considerations for Potential Applications of the AOP (optional)
At their discretion, the developer may include in this section discussion of the potential applications of an AOP to support regulatory decision-making. This may include, for example, possible utility for test guideline development or refinement, development of integrated testing and assessment approaches, development of (Q)SARs / or chemical profilers to facilitate the grouping of chemicals for subsequent read-across, screening level hazard assessments or even risk assessment. While it is challenging to foresee all potential regulatory application of AOPs and any application will ultimately lie within the purview of regulatory agencies, potential applications may be apparent as the AOP is being developed, particularly if it was initiated with a particular application in mind. This optional section is intended to provide the developer with an opportunity to suggest potential regulatory applications and describe his or her rationale. Detailing such considerations can aid the process of transforming narrative descriptions of AOPs into practical tools. In this context, it is necessarily beneficial to involve members of the regulatory risk assessment community on the development and assessment team. The Network view which is generated based on assessment of weight of evidence/degree of confidence in the hypothesized AOP taking into account the elements described in Section 7 provides a useful summary of relevant information as a basis to consider appropriate application in a regulatory context. Consideration of application needs then, to take into consideration the following rank ordered qualitative elements: Confidence in biological plausibility for each of the KERs Confidence in essentiality of the KEs Empirical support for each of the KERs and overall AOP The extent of weight of evidence/confidence in both these qualitative elements and that of the quantitative understanding for each of the KERs (e.g., is the MIE known, is quantitative understanding restricted to early or late key events) is also critical in determining appropriate application. For example, if the confidence and quantitative understanding of each KER in a hypothesised AOP are low and or low/moderate and the evidence for essentiality of KEs weak (Section 7), it might be considered as appropriate only for applications with less potential for impact (e.g., prioritisation, category formation for testing) versus those that have immediate implications potentially for risk management (e.g., in depth assessment). If confidence in quantitative understanding of late key events is high, this might be sufficient for an in depth assessment. The analysis supporting the Network view is also essential in identifying critical data gaps based on envisaged regulatory application. Instructions To edit the “Considerations for Potential Applications of the AOP” section, on an AOP page, in the upper right hand menu, click ‘Edit.’ This brings you to a page entitled, “Editing AOP.” Scroll down to the “Considerations for Potential Applications of the AOP” section, where a text entry box allows you to submit text. In the upper right hand menu, click ‘Update AOP’ to save your changes and return to the AOP page. The new text should appear under the “Considerations for Potential Applications of the AOP” section on the AOP page.
List the bibliographic references to original papers, books or other documents used to support the AOP. Instructions To edit the “References” section, on an AOP page, in the upper right hand menu, click ‘Edit.’ This brings you to a page entitled, “Editing AOP.” Scroll down to the “References” section, where a text entry box allows you to submit text. In the upper right hand menu, click ‘Update AOP’ to save your changes and return to the AOP page. The new text should appear under the “References” section on the AOP page.