title: The ability of the mouse lymphoma TK assay to detect aneugens.
abstract: There is some evidence that the mouse lymphoma TK assay (MLA) can detect aneugens, and this is accepted in the current International Conference on Harmonisation guidance for testing pharmaceuticals. However, whether or not it can be used as a reliable screen for aneugenicity has been the subject of debate. Consequently, aneugens with diverse mechanisms of action were tested in the MLA using 24-h exposure. No evidence of increased mutant frequency was seen with noscapine, diazepam or colchicine and increases were seen with taxol, carbendazim, econazole and chloral hydrate only at high levels of toxicity (for all but one taxol concentration survival reduced to ≤10% of control). None of these agents would be unequivocally classified as positive using currently accepted criteria. The largest increases in mutant number were seen with taxol and carbendazim; therefore, trifluorothymidine (TFT)-resistant clones resulting from treatment with them were cultured and analysed for chromosome 11 copy number using fluorescent in situ hybridisation (FISH) and loss of heterozygosity (LOH). High concentrations of these aneugens induced LOH at all loci examined indicating only one chromosome 11 was present but, perhaps surprisingly, all were found to have two copies of chromosome 11 using FISH. This would be consistent with loss of the tk(+) chromosome 11b with concomitant duplication of chromosome 11a, which has been proposed as a likely mechanism for induction of TFT-resistant clones. However, it was also surprising that analysis of centromere size showed that almost all the clones had both small and large centromeres, i.e. suggesting the presence of both chromosomes 11a and 11b. In conclusion, it appears that the TFT-resistant mutants resulting from treatment with toxic concentrations of some aneugens such as taxol and carbendazim have undergone complex genetic changes. However, these data show that the MLA cannot be used as a routine screen to detect aneugens.
authors.affiliation: AstraZeneca, R&D Alderley Park, Macclesfield, Cheshire SK10 4TG, UK. mick.fellows@astrazeneca.com
authors.forename: Michael D
authors.initials: MD
authors.lastname: Fellows
authors.forename: Ann T
authors.initials: AT
authors.lastname: Doherty
authors.forename: Catherine C
authors.initials: CC
authors.lastname: Priestley
authors.forename: Victoria
authors.initials: V
authors.lastname: Howarth
authors.forename: Michael R
authors.initials: MR
authors.lastname: O'Donovan
mesh_terms: D024422:Aneugens / Q000032:analysis* / Q000633:toxicity; D000818:Animals; D045744:Cell Line, Tumor; D002503:Centromere / Q000187:drug effects / Q000378:metabolism; D033481:Chromosomes, Mammalian / Q000235:genetics; D057075:Enzyme Assays / Q000379:methods*; D018628:Gene Dosage / Q000187:drug effects / Q000235:genetics; D017404:In Situ Hybridization, Fluorescence; D007621:Karyotyping; D019656:Loss of Heterozygosity / Q000187:drug effects / Q000235:genetics; D008223:Lymphoma / Q000378:metabolism*; D051379:Mice; D018895:Microsatellite Repeats / Q000235:genetics; D016133:Polymerase Chain Reaction; D013937:Thymidine Kinase / Q000378:metabolism*
chemical_list: D024422:Aneugens; D013937:Thymidine Kinase