Influence of the SCGE protocol on the amount of basal DNA damage detected in the Mediterranean mussel, Mytilus galloprovincialis.
Genotoxicity studies using the single cell gel electrophoresis (SCGE) assay indicate that basal levels of DNA strand breaks (SBs) in marine invertebrates are higher and more variable than those in marine vertebrates. This elevated level of DNA damage was attributed to a large number of alkali-labile sites, which are characteristic of the tightly-packaged DNA in invertebrate cells. To investigate if altering the SCGE protocol can artificially modulate high levels of SBs, SCGE experiments were performed on haemocytes from the Mediterranean mussel (Mytilus galloprovincialis) using proteinase K (PK) digestion in combination with assay buffers containing various concentrations of EDTA. In addition, the effects of Trolox (soluble antioxidant) and aurintricarboxylic acid (ATA; inhibitor of Ca(2+)/Mg(2+)-dependent nucleases) also were tested. The levels of SBs in M. galloprovincialis cells were compared with SBs in cells from a terrestrial mollusk (the snail Helix aspersa), and a teleost fish (the seabass Dicentrarchus labrax). The integrity of M. galloprovincialis DNA isolated with phenol extractions using EDTA, Trolox, and ATA was further assayed by gel electrophoresis. High SBs in mussel cells were reduced by combining EDTA with PK digestion, or using Trolox or ATA during cell processing for the SCGE assay. Snails and seabass had lower levels of SBs in the SCGE assay, and the levels were not affected by the protocol modifications. Adding EDTA, Trolox, or ATA to phenol extractions of M. galloprovincialis genomic DNA also reduced the extent of DNA fragmentation. These results suggest that the internal fluids of M. galloprovincialis may increase the basal levels of DNA SBs through oxidative and/or enzyme-mediated pathways. M. galloprovincialis is used extensively as a sentinel species for assessing the genotoxic hazard of marine pollutants. Our data suggest that the SCGE protocol should be carefully considered when assessing DNA damage in these species.
Machella
Nicola
N
Istituto di Biologia e Genetica, Università Politecnica delle Marche, Ancona, Italy. nm2272@columbia.edu
Battino
Maurizio
M
Pisanelli
Barbara
B
Regoli
Francesco
F
D000818:Animals; D001312:Aurintricarboxylic Acid / Q000494:pharmacology; D002021:Buffers; D002839:Chromans / Q000494:pharmacology; D020552:Comet Assay / Q000379:methods*; D053960:DNA Breaks; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000494:pharmacology; D019286:Endopeptidase K / Q000737:chemistry; D004784:Environmental Monitoring / Q000379:methods*; D006372:Helix, Snails / Q000235:genetics; D006434:Hemocytes / Q000187:drug effects; D009152:Mutagenicity Tests / Q000379:methods; D049878:Mytilus / Q000235:genetics*; D012680:Sensitivity and Specificity
D002021:Buffers; D002839:Chromans; D001312:Aurintricarboxylic Acid; D004492:Edetic Acid; D019286:Endopeptidase K; C010643:6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid