DNA integrity determination in marine invertebrates by Fast Micromethod

This study was focused toward the adaptation of the previously developed Fast Micromethod for DNA damage determination to marine invertebrates for the establishment of biomonitoring assessment. The Fast Micromethod detects DNA damage (strand breaks, alkali-labile sites and incomplete excision repair) and determines DNA integrity in cell suspensions or tissue homogenates in single microplates. The procedure is based on the ability of the specific fluorochrome dye PicoGreen to preferentially interact with high integrity DNA molecules, dsDNA, in the presence of ssDNA and proteins in high alkaline medium, thereby allowing direct fluorometric measurements of dsDNA denaturation without sample handling and stepwise DNA separations. The results presented herein describe the influence of the DNA amount and the pH of the denaturation media on slopes of the kinetic denaturation curves and calculated strand scission factors (SSFs). The optimal amount of DNA in Mytilus galloprovincialis gills homogenate was found to be 100 ng ml(-1) and the greatest differences in DNA unwinding kinetics (slopes and SSF values) were reached at pH 11.5. The induction of DNA damage and loss of DNA integrity was measured in native DNA isolated from cotton-spinner Holothuria tubulosa, marine sponge Suberites domuncula cells and mussel M. galloprovincialis gills homogenate. DNA damage and loss of DNA integrity were detected after induction by different doses of (gamma-rays, generated by 137Cs 1800 Ci; 0-500 rad in marine sponge S. domuncula cells up to SSFx(-1) values 0.082 +/- 0.012 for the highest radiation dose). Analysis by chemical xenobiotics based on the in vitro action of bleomycin (bleomycin-Fe(II) complex 0-50 or 0-83 microg ml(-1) (microM)) with native DNA from cotton-spinner H. tubulosa and mussel M. galloprovincialis gills homogenate yielded values of 0.537 +/- 0.072 and 0.130 +/- 0.018, respectively. In vivo experiments with mussel M. galloprovincialis gills homogenate by 4-nitroquinoline-N-oxide (NQO; 0-1 microg g(-1) NQO mussel) and benzo[a]pyrene (B[a]P; 0-20 microg g(-1) B[a]P mussel) indicated SSFx(-1) values of 0.121 +/- 0.016 and 0.090 +/- 0.007, respectively, for the highest applied doses of chemical xenobiotics. The analytical technique described here allows simple and fast analysis of DNA integrity, requires very short time for multiple analyses (less than 3 h) and even less than 100 ng DNA per single well (50 ng DNA isolated from cotton-spinner, 12,500 sponge cells or about 10 mg of mussel gills homogenate) in a microplate. This makes the Fast Micromethod applicable for the measurement of DNA integrity of small samples for genotoxicity assessment (biomonitoring), the effects of genotoxins on lower marine taxa or sessile invertebrates in marine environment (e.g. sponges, mussels) and the estimation of directional changes and harmful effects in the ecosystem.