Key Event Overview
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AOPs Including This Key Event
|AOP Name||Event Type||Essentiality|
|Chemical binding to tubulin in oocytes leading to aneuploid offspring||KE|
|Homo sapiens||Homo sapiens||Moderate||NCBI|
Level of Biological Organization
How this Key Event works
Microtubules are polar structures, and in each filament subunits are added to one extremity (the plus end) and removed from the other one (the minus end) [reviewed in Marchetti et al. 2015]. Microtubules are dynamic structures characterized by features such as dynamic instability and treadmilling. Dynamic instability defines the ability of microtubules to grow or shorten; the process is based on a multitude of events regulating the assembly/disassembly of the subunits. Treadmilling is the process by which, in the presence of an active loss of subunits (at the minus end) and acquisition of subunits (at the plus end), a steady-state is maintained and the length of the microtubule remains unchanged. Colchicine interferes with microtubule dynamics at lower concentrations while it induces a net depolymerization at higher concentrations which is a consequence of the inability of further extending the microtubules [Stanton et al. 2011]. This dual action is in common with other spindle poisons (e.g. vinca derivatives) [Panda et al. 1996]. All microtubule-binding agents alter microtubule dynamics, engaging cell cycle surveillance mechanisms that arrest cell division in metaphase. This mitotic stall may then lead to various irremediable effects such as mitotic catastrophe, apoptosis or aneuploidy [Kops et al. 2005].
How it is Measured or Detected
Microtubule depolymerization is generally assessed by an in vitro tubulin polymerization assay [Salmon et al. 1984; Wilson et al. 1984; Ibanez et al. 2003; Liu et al. 2010]. A reaction mixture containing tubulin and a test agent, after preincubation, is chilled on ice. GTP is added, and turbidity development is followed at 350 nm in a temperature controlled recording spectrophotometer. The extent of the reaction is then measured and the area under the curve is used to determine the concentration that inhibited tubulin polymerization by 50% (IC50) [Hamel 2003]. A concentration of 2.5 μM of colchicine is needed to inhibit microtubule polymerization by 50% [Zavala et al. 1980] and the ability of new chemicals to induce this effect is benchmarked against this value (e.g., combretastatin A-4 IC50 is 1.2 μM [Pettit et al. 1998]).
Consider the following criteria when describing each method: 1. Is the assay fit for purpose? Yes 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? Yes 3. Is the assay repeatable? Yes 4. Is the assay reproducible? Yes
Evidence Supporting Taxonomic Applicability
Depolymerization of microtubules has been measured in many somatic cell types, in addition to frog and mouse eggs, and in human cells, including eggs, in culture [Salmon et al. 1984; Wilson et al. 1984; Ibanez et al. 2003; Liu et al. 2010].
Hamel E. 2003. Evaluation of antimitotic agents by quantitative comparisons of their effects on the polymerization of purified tubulin. Cell Biochem Biophys 38:1-22.
Ibanez E, Albertini DF, Overstrom EW. 2003. Demecolcine-induced oocyte enucleation for somatic cell cloning: coordination between cell-cycle egress, kinetics of cortical cytoskeletal interactions, and second polar body extrusion. Biol Reprod 68:1249-1258.
Kops GJ, Weaver BA, Cleveland DW. 2005. On the road to cancer: aneuploidy and the mitotic checkpoint. Nat Rev Cancer 5:773-785.
Liu S, Li Y, Feng HL, Yan JH, Li M, Ma SY, Chen ZJ. 2010. Dynamic modulation of cytoskeleton during in vitro maturation in human oocytes. Am J Obstet Gynecol 203:151.e151-157.
Marchetti A, Massarotti A, Yauk CL, Pacchierotti F, Russo A. Submitted. The adverse outcome pathway (AOP) for chemical binding to tubulin in oocytes leading to aneuploid offspring. Environ Mol Mutagen.
Panda D, Jordan MA, Chu KC, Wilson L. 1996. Differential effects of vinblastine on polymerization and dynamics at opposite microtubule ends. J Biol Chem 271:29807-29812.
Pettit GR, Toki B, Herald DL, Verdier-Pinard P, Boyd MR, Hamel E, Pettit RK. 1998. Antineoplastic agents. 379. Synthesis of phenstatin phosphate. J Med Chem 41:1688-1695.
Salmon ED, McKeel M, Hays T. 1984. Rapid rate of tubulin dissociation from microtubules in the mitotic spindle in vivo measured by blocking polymerization with colchicine. J Cell Biol 99:1066-1075.
Stanton RA, Gernert KM, Nettles JH, Aneja R. 2011. Drugs that target dynamic microtubules: a new molecular perspective. Med Res Rev 31:443-481.
Wilson L, Miller HP, Pfeffer TA, Sullivan KF, Detrich HW, 3rd. 1984. Colchicine-binding activity distinguishes sea urchin egg and outer doublet tubulins. J Cell Biol 99:37-41.
Zavala F, Guenard D, Robin JP, Brown E. 1980. Structure--antitubulin activity relationship in steganacin congeners and analogues. Inhibition of tubulin polymerization in vitro by (+/-)-isodeoxypodophyllotoxin. J Med Chem 23:546-549.