The DNA repair inhibitors hydroxyurea and cytosine arabinoside enhance the sensitivity 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 electrophoresis ('comet') assay. We therefore sought to increase its sensitivity by inhibiting DNA repair during exposure to test compounds, using 10 mM hydroxyurea (HU) and 1.8 mM cytosine arabinoside (ara-C), which inhibit DNA resynthesis during nucleotide excision repair. The following compounds were tested, using a 30-min exposure, in the absence or presence of HU/ara-C: 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4, 8-DiMeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (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]pyridine (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 background level of comet formation was reasonably constant over several months and was increased only slightly, but significantly, in the presence of the DNA-repair inhibitors. All compounds that induced comet formation did so without appreciable cytotoxicity as assessed by trypan blue exclusion. Of the compounds tested, the heterocyclic amines and polycyclic aromatic hydrocarbons (with the exceptions of PhIP and B[a]P) failed to induce convincing levels of comet formation in the absence of repair inhibitors. In their presence the heterocyclic amines tested induced comet formation (with the exception of 8-MeIQx), with widely differing potencies. 1-NP failed to elicit marked 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, lindane, bleomycin, cisplatin, MNNG, sodium chromate and chromic chloride induced 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 more agents need to be tested in order to determine how well the comet assay using MCL-5 cells (or modified versions of it) can distinguish genotoxins from non-genotoxins.
Martin
F L
FL
Institute of Cancer Research, Haddow Laboratories, Cotswold Rd., Sutton, UK. flmartin@icr.ac.uk
Cole
K J
KJ
Orme
M H
MH
Grover
P L
PL
Phillips
D H
DH
Venitt
S
S
D000588:Amines / Q000633:toxicity; D001554:Benzene / Q000633:toxicity; D001761:Bleomycin / Q000633:toxicity; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002712:Chlorides / Q000633:toxicity; D002840:Chromates / Q000633:toxicity; D017608:Chromium Compounds / Q000633:toxicity; D002945:Cisplatin / Q000633:toxicity; D003561:Cytarabine / Q000494:pharmacology*; D004247:DNA / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair / Q000187:drug effects*; D004054:Diethylstilbestrol / Q000633:toxicity; D004305:Dose-Response Relationship, Drug; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D006571:Heterocyclic Compounds / Q000633:toxicity; D001556:Hexachlorocyclohexane / Q000633:toxicity; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology*; D008769:Methylnitronitrosoguanidine / Q000633:toxicity; D009152:Mutagenicity Tests; D009574:Nitro Compounds / Q000633:toxicity; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology*; D011084:Polycyclic Aromatic Hydrocarbons / Q000633:toxicity; D011839:Radiation, Ionizing; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017670:Sodium Compounds / Q000633:toxicity; D013395:Sucrose / Q000494:pharmacology
D000588:Amines; D002712:Chlorides; D002840:Chromates; D017608:Chromium Compounds; D006571:Heterocyclic Compounds; D009574:Nitro Compounds; D019384:Nucleic Acid Synthesis Inhibitors; D011084:Polycyclic Aromatic Hydrocarbons; D017670:Sodium Compounds; D003561:Cytarabine; D001761:Bleomycin; D008769:Methylnitronitrosoguanidine; D013395:Sucrose; D001556:Hexachlorocyclohexane; C028982:sodium chromate(VI); D004054:Diethylstilbestrol; D004247:DNA; C017133:chromous chloride; D001554:Benzene; D002945:Cisplatin; D006918:Hydroxyurea