The DNA repair inhibitors hydroxyurea and cytosine arabinoside enhance thesensitivity of the alkaline single-cell gel electrophoresis ('comet') assay in metabolically-competent MCL-5 cells

We have found previously that the metabolically-competent human MCL-5 cell line did not appear to be usefully sensitive to the DNA-damaging effects of several carcinogens, as measured by the alkaline single-cell gel electroph oresis ('comet') assay. We therefore sought to increase its sensitivity by inhibiting DNA repair during exposure to test compounds, using 10 mM hydrox yurea (HU) and 1.8 mM cytosine arabinoside (ara-C), which inhibit DNA resyn thesis during nucleotide excision repair. The following compounds were test ed, using a 30-min exposure, in the absence or presence of HU/ara-C: 2-amin o-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), 2-amino-3,4,8-trimethyli midazo[4,5-f]quinoxaline (4,8-DiMeIQx), 2-amino-3-methylimidazo[4,5-f]quino line (IQ), 2-amino-9H-pyrido[2,3-b]indole (A[alpha]C), 2-amino-3-methyl-9H- pyrido[2,3-b]indole (MeA[alpha]C), 2-amino-1-methyl-6-phenylimidazo[4,5-b]p yridine (PhIP), benzo[a]pyrene (B[a]P), 3-methylcholanthrene (3-MCA), 7,12- dimethylbenz[a]anthracene (DMBA), 1-nitropyrene (1-NP), 2-nitrofluorene (2- NF), aniline, o-toluidine, benzene, lindane, bleomycin, cisplatin, N-methyl -N'-nitro-N-nitrosoguanidine (MNNG), sodium chromate, chromic chloride, and diethylstilboestrol (DES). We made the following observations. The backgro und level of comet formation was reasonably constant over several months an d was increased only slightly, but significantly, in the presence of the DN A-repair inhibitors. All compounds that induced comet formation did so with out appreciable cytotoxicity as assessed by trypan blue exclusion. Of the c ompounds tested, the heterocyclic amines and polycyclic aromatic hydrocarbo ns (with the exceptions of PhIP and B[a]P) failed to induce convincing leve ls of comet formation in the absence of repair inhibitors. In their presenc e the heterocyclic amines tested induced comet formation (with the exceptio n of 8-MeIQx), with widely differing potencies. 1-NP failed to elicit marke d comet formation even in the presence of HU/ara-C. Aniline and o-toluidine produced significant levels of comet formation in the absence of HU/ara-C, but in their presence comet formation was markedly increased. Benzene, lin dane, bleomycin, cisplatin, MNNG, sodium chromate and chromic chloride indu ced comet formation in the absence of HU/ara-C, but, with the exception of cisplatin, their presence enhanced comet formation. Neither sucrose nor DES elicited comet formation under the conditions used in this study. Many mor e agents need to be tested in order to determine how well the comet assay u sing MCL-5 cells (or modified versions of it) can distinguish genotoxins fr om non-genotoxins. (C) 1999 Elsevier Science B.V. All rights reserved.