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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 Health Protection Technology Division, Laboratory of Biosafety and Risk Assessment, ENEA CR Casaccia, Rome, Italy. 4 Department of Molecular Medicine, University of Padova, Padova, Italy.
- Correspondence to: Francesco Marchetti, Environmental Health Science and Research Bureau, Health Canada, Ottawa, ON K1A 0K9, Canada. E-mail: email@example.com
Point of Contact
Francesco Marchetti (email 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 Do not cite||Under Development|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 June 02, 2019 19:30
|Binding, Tubulin||December 11, 2018 15:15|
|Disruption, Microtubule dynamics||May 27, 2019 14:10|
|Disorganization, Spindle||May 27, 2019 14:15|
|Increase, Aneuploid offspring||May 27, 2019 14:54|
|Altered, Meiotic chromosome dynamics||May 27, 2019 14:17|
|Altered, Chromosome number||May 27, 2019 14:29|
|Binding, Tubulin leads to Disruption, Microtubule dynamics||May 27, 2019 15:06|
|Disruption, Microtubule dynamics leads to Disorganization, Spindle||May 27, 2019 15:12|
|Disorganization, Spindle leads to Altered, Meiotic chromosome dynamics||May 27, 2019 15:25|
|Altered, Chromosome number leads to Increase, Aneuploid offspring||May 27, 2019 15:15|
|Binding, Tubulin leads to Altered, Chromosome number||May 27, 2019 15:19|
|Altered, Meiotic chromosome dynamics leads to Altered, Chromosome number||May 27, 2019 15:30|
|Colchicine||November 29, 2016 18:42|
|Vinblastine sulfate||May 27, 2019 15:42|
|Benomyl||November 29, 2016 18:42|
|Nocodazole||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 animal studies, there is also one reported case in which environmental exposure to trichlorfon, an organophosphate insecticide, was associated with a cluster of Down syndrome cases among women in a 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 depolymerization leads to meiotic spindle disorganization (KE2), which in turns lead to altered chromosome dynamics (KE3) and the generation of aneuploid oocytes (KE4). Aneuploid oocytes can be fertilized and generate aneuploid 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; Webster and Schuh, 2017]. 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; Webster and Schuh, 2017], 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].
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 [Hunt and Hassold, 2002; Nagaoka et al., 2012]. The prevalence of chromosome segregation errors during female meiosis is clearly supported by the application of state-of-the-art genomic approaches, such as Comparative Genomic Hybridization (CGH), array-Comparative Genomic Hybridization (aCGH), SNP-arrays [Handyside, 2012; Nagaoka et al., 2012] and next generation sequencing (NGS) [Hou et al., 2013; Kung et al., 2015; Treff et al., 2016].
The present AOP focuses on chemical binding to tubulin that causes depolymerization of microtubules and generation of aneuploid cells. Although this molecular initiating event can occur in any cell, the adverse outcome is the generation of aneuploid conceptuses; therefore, this AOP is specific to germ cells, and in particular, to female germ cells.
Gianaroli L, Magli MC, Cavallini G, Crippa A, Capoti A, Resta S, Robles F, Ferraretti AP. 2010. Predicting aneuploidy in human oocytes: key factors which affect the meiotic process. Hum Reprod 25:2374-2386.
Handyside AH. 2012. Molecular origin of female meiotic aneuploidies. Biochim Biophys Acta 1822:1913-1920.
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.
Hou Y, Fan W, Yan L, Li R, Lian Y, Huang J, Xu L, Tand F, Xiw XS, Qiao J. 2013. Genome analyses of single human oocytes. Cell 155:1492-1506.
Hunt PA, Hassold TJ. 2002. Sex matters in meiosis, Science 296:2181-2183.
Kuliev A, Cieslak J, Ilkevitch Y, Verlinsky Y. 2003. Chromosomal abnormalities in a series of 6,733 human oocytes in preimplantation diagnosis for age-related aneuploidies. Reprod Biomed Online 6:54-59.
Kung A, Munné S, Bankowski B, Coates A, Wells D. 2015. Validation of next-generation sequencing for comprehensive chromosome screening of embryos. Reprod Biomed Online 31:760-769.
Magli MC, Gianaroli L, Ferraretti AP. 2001. Chromosomal abnormalities in embryos. Mol Cell Endocrinol 183:S29-34.
Munne S. 2002. Preimplantation genetic diagnosis of numerical and structural chromosome abnormalities. Reprod Biomed Online 4:183-196.
Nagaoka SI, Hassold TJ, Hunt PA. 2012. Human aneuploidy: Mechanisms and new insights into an age-old problem. Nat Rev Genet 13:493–504.
Nicolaidis P, Petersen MB. 1998. Origin and mechanisms of non-disjunction in human autosomal trisomies. Hum Reprod 13:313-319.
Treff NR, Kirsher RL, Tao X, Garnsey H, Boher C, Silva E, Landis J, Taylor D, Scott RT, Woodruff TK, Duncan FE. 2016. Next Generation Sequencing-based comprehensive chromosome screening in mouse polar bodies, oocytes, and embryos. Biol Reprod 94:76
Webster A, Schuh M. 2017. Mechanisms of aneuploidy in human eggs. Trends Cell Biol 27:55-68.
Summary of the AOP
Events: Molecular Initiating Events (MIE)
|Sequence||Type||Event ID||Title||Short name|
|1||MIE||718||Binding, Tubulin||Binding, Tubulin|
|2||KE||720||Disruption, Microtubule dynamics||Disruption, Microtubule dynamics|
|3||KE||721||Disorganization, Spindle||Disorganization, Spindle|
|4||KE||752||Altered, Meiotic chromosome dynamics||Altered, Meiotic chromosome dynamics|
|5||KE||723||Altered, Chromosome number||Altered, Chromosome number|
|6||AO||728||Increase, Aneuploid offspring||Increase, Aneuploid offspring|
Relationships Between Two Key Events
(Including MIEs and AOs)
|Binding, Tubulin leads to Altered, Chromosome number||non-adjacent||High|
Life Stage Applicability
|Adult, reproductively mature||High|
|Mus musculus||Mus musculus||High||NCBI|
|Homo sapiens||Homo sapiens||Moderate||NCBI|
Overall Assessment of the AOP
A comprehensive review of the literature (Supplementary Table 1) was conducted to gather the available studies in which the effects of microtubule inhibitors were tested for the induction of aneuploidy in female germ cells. The focus of this AOP is on spindle poisons that bind to tubulin resulting in microtubule depolymerization leading to abnormalities in spindle function and chromosomal dynamic ultimately resulting in an egg with an abnormal number of chromosomes. Although chemicals with different mechanism of actions, such as topoisomerase II inhibitors, also have strong data showing aneugenic activity in female germ cells [Mailhes and Marchetti, 2005; Pacchierotti et al., 2007], chemicals that bind to tubulin represent the largest class for which the aneugenic activity has been evaluated [Marchetti et al., 2016]. Studies providing sufficient information regarding doses, timing of exposure and egg collection, and experimental results were considered to assess the empirical data supporting each of the KEs and KERs. It should be noted that, as mentioned before, few studies have investigated multiple KEs within the same study design and the majority of the data available refers to the induction of aneuploidy. An additional complication is that the MIE and first KE (microtubule depolymerization) are most often assessed in acellular systems rendering the quantitative assessment of the concordance among upstream KEs and downstream KEs complex. However, those few cases where multiple KEs were investigated showed concordance for both dose-related and time-related effects [Shen et al., 2005; Eichenlaub-Ritter et al., 2007]. As a whole, we consider the studies in Supplementary Table 1 to provide extensive and convincing evidence that tubulin-binding chemicals cause microtubule depolymerization and spindle disturbances leading to the generation of aneuploid eggs. Strong in vivo dose-response data on the induction of aneuploid eggs is available for several chemicals, including colchicine, benomyl, and vinblastine [reviewed in Mailhes and Marchetti, 1994; 2005; Pacchierotti et al., 2007]. Data with colchicine is also available to demonstrate that aneuploid eggs are fertilized and that the frequencies of aneuploidy are similar before and after fertilization [Mailhes et al., 1990]. Overall, we consider that the available data provide high support for this AOP as a whole, while empirical support for the different KERs is varied.
Domain of Applicability
The present AOP should be considered specific to female germ cells exposed in the peri-ovulation period. The majority of data in this AOP were derived from experiments in mice, however, relevant endpoints have been evaluated in a variety of higher and lower eukaryotes. The available results on the induction of aneuploidy 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. In addition, the similarities in oogenesis between rodents and humans suggest 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 show an increase in the occurrence of high levels of aneuploid oocytes [Leland et al., 2009; McGuinness et al., 2009; reviewed in Mailhes and Marchetti, 2010].
The final step of the AOP requires the transmission of the aneuploid condition from the oocyte to the offspring. Since the available data suggest that there is a narrow window of sensitivity for the induction of aneuploidy by spindle poisons (around the time of resumption of meiosis in preparation for ovulation) it could be possible to wait longer periods of time after the administration of colchicine, or any other of the chemicals listed in this AOP, and demonstrate that under these conditions there is no transmission of aneuploidy to the offspring. However, no such study has been conducted.
Biological plausibility of the KERs is strong. There is clear understanding of the MIE for many of the chemicals listed in this AOP. Both the colchicine- and vinca alkaloid-binding sites on tubulin are characterized in detail. The consequences of chemical binding to tubulin for microtubule dynamics are also qualitatively and quantitatively well understood. It also broadly accepted that microtubule dynamics is essential for proper spindle assembly and function. There is less understanding of why the SAC is unable to prevent meiotic progression in the presence of misaligned chromosomes [Marchetti et al., 2006; Webster and Schuh, 2017] and this represent a key research gap.
Empirical support for the KERs is generally strong, although the empirical evidence and our understanding of the KER between abnormalities in chromosome dynamics and generation of an aneuploidy egg is limited. The strongest empirical support is associated with the indirect KER linking binding of chemicals to tubulin with the induction of aneuploid eggs. 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., 1993; Sun et al., 2005; Eichenlaub-Ritter et al., 2007].
As described in the previous sections of the AOP, it is well established that chemicals that bind to tubulin affect the polymerization of microtubules triggering abnormalities in the meiotic spindle and the subsequent chromosomal missegregation. There is also sufficient evidence to show that these events increase with dose in a manner that is consistent with this AOP. Binding to tubulin seems to increase linearly with dose, however, microtubule depolymerization must exceed a threshold before abnormalities in the meiotic spindles become apparent. There is also sufficient evidence that there is a threshold for the induction of aneuploidy. However, the precise quantitative relationship has not been established and it may be different for different chemicals. This is because different chemicals may induce different degrees of arrest at the metaphase of the first meiotic division which would prevent the manifestation of the aneuploidy in metaphase II oocytes.
Considerations for Potential Applications of the AOP (optional)
There are no established OECD test guidelines (TG) for measuring aneuploidy in oocytes. However, there are several existing TGs, such as the in vivo and in vitro micronucleus test (OECD TG 474 and OECD TG 487) and the in vivo and in vitro chromosomal aberration test (OECD TG 475 and OECD TG 473), and one specific to spermatogonial cells (OECD TG 483) that although not specifically designed to detect aneuploidy can provide evidence of aneugenic activity. Although it is generally assumed that data obtained in somatic cells can be extrapolated to germ cells to inform regulatory decisions, the availability of germ cell data is critical for the proper classification of products under the Globally Harmonized System (GHS) of classification and labelling [United Nations, 2013]. In addition, the recent International Workshops on Genotoxicity Testing that took place in Tokyo, Japan in November 2017 included a workgroup that addressed the risk of aneugens for human health assessment. As part of the work, the group reviewed all available data for germ cell aneugens in mammals, independently of the mechanism of action. Therefore, the present AOP addresses at topic of high interest among the genotoxicity community and may help in identifying research gaps and direct future work.
Eichenlaub-Ritter U, Winterscheidt U, Vogt E, Shen Y, Tinneberg HR, Sorensen R. 2007. 2-methoxyestradiol induces spindle aberrations, chromosome congression failure, and nondisjunction in mouse oocytes. Biol Reprod 76:784–793.
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, 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.
Mailhes JB, Marchetti F. 2010. Advances in understanding the genetic causes and mechanisms of female germ cell aneuploidy. Exp Rev Obst Gyn 5:687–706.
Mailhes JB, Aardema MJ, Marchetti F. 1993. Investigation of aneuploidy induction in mouse oocytes following exposure to vinblastine-sulfate, pyrimethamine, diethylstilbestrol diphosphate, or chloral hydrate. Environ Mol Mutagen 22:107–114.
Mailhes JB, Carabatsos MJ, Young D, London SN, Bell M, Albertini DF. 1999. Taxol-induced meiotic maturation delay, spindle defects, and aneuploidy in mouse oocytes and zygotes. Mutat Res 423:79-90
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.
McGuinness BE, Anger M, Kouznetsova A, Gil-Bernabé AM, Helmhart W, Kudo NR, Wuensche A, Taylor S, Hoog C, Novak B. Nasmyth K. 2009. Regulation of APC/C activity in oocytes by a Bub1-dependent spindle assembly checkpoint. Curr Biol 19:369-380.
OECD. 2016. Test No. 473: In Vitro Mammalian Chromosomal Aberration Test, OECD Publishing, Paris. http://dx.doi.org/10.1787/9789264264649-en.
OECD. 2016. Test No. 474: Mammalian Erythrocyte Micronucleus Test, OECD Publishing, Paris. http://dx.doi.org/10.1787/9789264264762-en.
OECD. 2016. Test No. 475: Mammalian Bone Marrow Chromosomal Aberration Test, OECD Publishing, Paris. http://dx.doi.org/10.1787/9789264264786-en.
OECD. 2016. Test No. 483: Mammalian Spermatogonial Chromosomal Aberration Test, OECD Publishing, Paris. http://dx.doi.org/10.1787/9789264264786-en.
OECD. 2016. Test No. 487: In Vitro Mammalian Cell Micronucleus Test, OECD Publishing, Paris. http://dx.doi.org/10.1787/9789264264861-en.
Ou XH, Li S, Xu BZ, Wang ZB, Quan S, Li M, Zhang QH, Ouyang YC, Schatten H, Xing FQ, Sun QY. 2010. p38α MAPK is a MTOC-associated protein regulating spindle assembly, spindle length and accurate chromosome segregation during mouse oocyte meiotic maturation. Cell Cycle 9:4130-4143.
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.
Russo A, Pacchierotti F. 1988. Meiotic arrest and aneuploidy induced by vinblastine in mouse oocytes. Mutat Res 202:215–221.
Shen Y, Betzendahl I, Sun F, Tinneberg HR, Eichenlaub-Ritter U. 2005. Non-invasive method to assess genotoxicity of nocodazole interfering with spindle formation in mammalian oocytes. Reprod Toxicol 19:459–471.
Sun F, Betzendahl I, Pacchierotti F, Ranaldi R, Smitz J, Cortvrindt R, Eichenlaub-Ritter U. 2005. Aneuploidy in mouse metaphase II oocytes exposed in vivo and in vitro in preantral follicle culture to nocodazole. Mutagenesis 20:65–75.
United Nations 2013. Globally Harmonized System of Classification and Labelling of Chemicals (GHS), Fifth revised edition ed., New York and Geneva.
Webster A, Schuh M. 2017. Mechanisms of aneuploidy in human eggs. Trends Cell Biol 27:55-68