Comparative evaluation of the alkaline comet assay with the micronucleus test for genotoxicity monitoring using aquatic organisms

A comparative analysis between the in vivo comet assay and the in vivo micronucleus test (MNT) was carried out in three aquatic organisms suitable for genotoxicity monitoring, carp (Cyprinus carpio), rainbow trout (Oncorhynchus mykiss), and clam (Spisula sachalinensis), using a direct-acting mutagen, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), and an indirect mutagen, benzo[a]pyrene (B[a]P). By optimizing the conditions for cell isolation, gill and liver (or digestive glands) were selected as test tissues of the comet assay for MNNG and B[a  ]P. The MNT employed the erythrocytes (or hemocytes), the most universal cell type for the assay. The analysis of DNA strand breaks using the comet assay and the micronucleus frequencies using the MNT revealed dose- and time-dependent increases between animals exposed to several concentrations of mutagens. But the statistical significance (P<0.05P<0.05) obtained was higher by the comet assay than by the MNT. When the time profiles of genotoxic signals resulting from B[a]P exposure to carp were plotted representatively, clear distinctions between all concentrations were made in the comet assay, but not in the MNT. The correlation index defined in this study also showed a higher correlation between concentration and signal in the comet assay than in the MNT. It is suggested that the standardization of the comet assay is necessary for its methodological evaluation and use as a genotoxicity biomarker. We conclude that the comet assay has an excellent suitability for aquatic genotoxicity monitoring because of its high and reliable sensitivity.