Effects of 10 known or suspected spindle poisons in the in vitro porcine brain tubulin assembly assay

We tested the 10 known or suspected spindle poisons (colchicine, econazole nitrate, chloral hydrate, hydroquinone, diazepam, thiabendazole, cadmium chloride, thimerosal, pyrimethamine and vinblastine) of the coordinated EEC programme for induction of aneuploidy with the in vitro porcine brain tubulin assembly assay. The influence of the compounds on different parameters [lag-phase, polymerization velocity, endabsorption (steady-state level), reversibility, influence on disassembly at 4°C] was evaluated. Colchicine [IC30 (30% inhibition concentration): 0.002 mM), vinblastine (IC30: 0.002 mM), thimerosal (IC30: 0.03 mM), thiaben-dazole (IC30: 0.5 mM) and chloral hydrate (IC30: 60 mM) led to an inhibition of tubulin assembly in vitro. No influence on the steady-state level was obtained with econazole nitrate (up to 0.1 mM), diazepam (up to 2.5 mM), cadmium chloride (up to 1 mM), pyrimethamine (up to 1 mM) and hydroquinone (up to 25 mM), the highest dose tested being limited either by precipitation or by reaching the maximal solubility of the compound in the solvent used. Diazepam enhanced the lag-phase and slightly reduced the polymerization velocity dose-dependently; however, all the treated test mixtures reached the same end absorption levels as the control. The influence on the disassembly process was studied at 4°C. Microtubules treated with colchicine, econazole nitrate, diazepam, thiabendazole, cadmium chloride, thimerosal and pyrimethamine reached the same end absorption level after disassembly as the untreated control. Chloral hydrate reduced the disassembly rate but the end absorption of the control was not reached, the 30% reduction concentration being 0.25 mM. Hydroquinone at very high doses (>10mM) stimulated the disassembly process. The in vitro tubulin assembly assay is, on the basis of this small database, an indicative pre-screening test for aneuploidy inducing chemicals which act mechanistically via interaction with tubulin and/or microtubule associated proteins, both components of the spindle apparatu