| An in vitro assay system for detecting cellular DNA damage in FKM (fathead minnow) exposed to genotoxicants, HN2 (nitrogen mustard) and MNNG (N-methyl,N{dollar}spprime{dollar}-nitro-N-nitrosoguanidine), is described. DNA from the exposed FHM was examined for cellular DNA strand breakage by agarose gel electrophoresis. FHM were exposed to a series of nitrogen mustard concentrations (0, 10, 20, 30, 50, 100, 200, 500 mg/L) dissolved in MHW (moderately hard water) for 2 hr. The DNA was isolated, purified and checked with the spectrophotometer so that the OD260/OD280 ratio was not less than 1.6. DNA samples were then separated by 0.7% agarose gel electrophoresis for 2 hr at a constant voltage of 50. The gel was then photographed with a IS-1000 Digital Imaging System. The cellular DNA bands were identified and quantified with the ImageQuaNT{dollar}rmsp{lcub}TM{rcub}{dollar} software. The control group's DNA content, set at 100%, was compared to the DNA derived from various HN2 exposures. A dose-dependent response curve based on DNA content was observed from which an EC50 value was derived. Similarly, the MNNG exposure study performed at comparable concentrations yielded a dose-dependent curve based on DNA content. Samples from a wastewater treatment plant and a metal planting company were utilized to demonstrate for the DNA damage assay in fathead minnows. This assay proved to be a more sensitive indicator for evaluating aquatic pollutant when compared to the standard acute toxicity routinely employed. Based on these laboratory data, it appears that this in vitro assay could have applicability in screening for aquatic genotoxic pollutant in the environment. The relationships between acute (LC50) and chronic endpoints of aquatic organisms and this in vitro assay were investigated and discussed. |
