Establishment Of A Multiple-Endpoint System For Genotoxicity Test Based On Human Cells

Both the accurate assessment on genotoxicity and profound analysis on mechanism of exobiotics have been key research issues in the field of toxicology all along. Our study aims at establishing a multiple-endpoint system for genotoxicity test based on human cells including comet assay, in vitro micronucleus test and tk gene mutation analysis, evaluating the genotoxicity of three chemicals with different mechanism, namely methyl methanesulfonate (MMS), mitomycin C (MMC) and sodium azide (NaN3), and providing data for the further use of the test system in rapid screening for genotoxicity of chemicals, and raw materials of food and drugs.In comet assay, both the percentage of comet cells and the length of comet tail in WTK1 cell line and human peripheral lymphocytes (HPL) increased significantly at two different sampling time induced by MMS at concentration from 0.625 to 5 μg/ml. The inducing effect on both cell types of MMC at concentration from 0.0625 to 0.5 μg/ml was observed at 20 hours but not immediately after exposure, while NaN3 showed its effect on the two cell types only at concentration of 2000 μg/ml at different sampling time. The effect of MMS and NaN3 was weaker at 20 hours compared with that immediately after exposure.In in vitro micronucleus test, both MMS and MMC induced increase of micronucleated cell (MNC) frequency in the two cell types at 20 and 44 hours after exposure, but NaN3 showed no obvious inducing effect. All the three test chemicals induced higher MNC frequency in WTK1 cell line at 44 hours.while in HPL, MNC frequency induced by MMS and MMC was higher at 20 hours. The effect of NaN3 in HPL was similar at the two sampling time.In tk gene mutation assay, all the three chemicals induced tk gene mutation. A majority of the trifluorothymidine-resistant mutants induced by three chemicals were slow-growth colonies, all the colonies analyzed had lost one or more exon(s), and most of which had lost exon 4 and exon 5-7 detected by multiplex PCR. Obvious hotspot existed in WTK1 cell tk gene mutation.The results of this study showed that MMS and MMC could induce gene mutation, DNA single strand break (SSB) and chromosome aberration (CA) in WTK1 cell line and HPL, indicating their strong genotoxicity. NaN3 could induce tk gene mutation, but had no obvious effect in inducing CA, and caused SSB only at the highest concentration, indicating its potential genotoxicity. The study also suggested that it was convenient and feasible to use the same culture of WTK1 cell line to test multiple endpoints such as DNA damage, chromosome aberration and gene mutation. So it was well worth popularizing and applying to rapid screening of mutagens and carcinogens.