Genotoxic effects of selected biocides on RTG-2 fish cells by means of a modified Fast Micromethod Assay

A sensitive in vitro assay for detecting DNA damage in RTG-2 cells culture is described. This assay employs a dye, PicoGreen double stranded DNA (dsDNA) quantitation reagent, which becomes intensely fluorescent upon binding nucleic acids. The assay includes a simple and rapid 50-min sample lysis in the presence of EDTA, SDS, and high urea concentration at pH 10, followed by time-dependent DNA denaturation at pH 11.6 after NaOH addition. The time course and the extent of DNA denaturation are followed in a microplate fluoresecence reader at room temperature for less than 1 h. Comparative studies between suspension and fixed RTG-2 cells indicated that it is possible to apply this methodology in both cases with good results. Neutral red assay was used for to determine the cellular viability when RTG-2 cultures were exposed to tetrakis(hydroxymethyl) phosphonium chloride (THPC) and benzalkonium chloride (BC), as biocides used in the disinfection of cooling towers. The results obtained by neutral red assay indicate IC50(48) values of 0.017 (0.011-0.028) and 2.71 (1.91-3.86) mg/L for tetrakis(hydroxymethyl) phosphonium chloride and benzalkonium chloride, respectively. DNA damage has been evaluated for both disinfectants in RTG-2 culture, by exposure to 1/10-, 1/25-, 1/50-, and 1/100-IC50(48) value, and the results obtained indicate a strain scission factor (SSF) of 0.126 ± 0.014, 0.181 ± 0.014, 0.217 ± 0.013, and 0.245 ± 0.013 in cell suspensions, and 0.077 ± 0.019, 0.107 ± 0.014, 0.151 ± 0.014, and 0.202 ± 0.015 in attached cells for tetrakis(hydroxymethyl) phosphonium chloride; while the SSF values for benzalkonium chloride are 0.023 ± 0.009, 0.033 ± 0.017, 0.068 ± 0.012, and 0.088 ± 0.015 in cell suspensions, and 0.033 ± 0.010, 0.044 ± 0.011, 0.080 ± 0.009, and 0.093 ± 0.010 in attached cells. Thus, the assay proposed in this study has made it possible to show DNA damage in RTG-2 cells when exposed to 0.2 (1/100 IC50(48)) and 300 (1/10 IC50(48)) Hg/L of tetrakis(hydroxymethyl) phosphonium chloride and benzalkonium chloride, respectively. The results obtained indicate that the Fast Micromethod Assay, applied on RTG-2 cell line cultures, is a fast and sensitive method for the early DNA damage detection in the aquatic environment.