Influence of the SCGE protocol on the amount of basal DNA damage detected in the Mediterranean mussel, Mytilus galloprovincialis

Abstract

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.

An ecotoxicological protocol with caged mussels, Mytilus galloprovincialis, for monitoring the impact of an offshore platform in the Adriatic sea

“…Genotoxic biomarkers in sentinel species, are recommended in monitoring investigations and both the Comet assay and the micronucleus test have been largely validated in Mediterranean mussels (Frenzilli et al, 2001;Regoli et al, 2004;Machella et al, 2006;Nigro et al, 2006). In our study, the Comet assay demonstrated a moderate increase of DNA fragmentation in mussels caged to the platform during the first period, further supporting the slight oxidative challenge revealed by antioxidant responses and lysosomal membrane stability.…”

“…The comet assay was carried out on haemocytes immediately diluted in Ca 2+ /Mg 2+ -free buffer, centrifuged, resuspended in 0.6% low-melting-point agarose, and included in 1% normal-melting-point agarose on glass slides (Machella et al, 2006). Slides were placed into the lysing solution (2.5 M NaCl, 100 mM EDTA, 1% Triton X-100, and 10% DMSO, pH 10, 4°C in the dark for 90 min), and the DNA was unwound in 75 mM NaOH, 10 mM EDTA (pH 13) before the electrophoresis (1 V/cm for 10 min).…”

Comet assay: a reliable tool for the assessment of DNA damage in different models

“…Also, seasonal variation and temperature altered both DNA damage baseline levels in untreated animals and cell sensitivity towards environmental pollutants under in vitro conditions (Buschini et al 2003;Hartl et al 2004). The Comet assay detecting DNA strand breaks has demonstrated that higher basal levels of DNA damage are observed in marine invertebrates; hence, the protocol followed in these animals should be considered for biomonitoring the ecogenotoxicity of a region (Machella et al 2006).…”

Seasonal variations of exposure biomarkers, oxidative stress responses and cell damage in the clams, Tapes philippinarum, and mussels, Mytilus galloprovincialis, from Adriatic sea

“…Haemolymph collected from ten specimens was immediately processed for LMS and DNA integrity. Metallothioneins-like proteins (MTSH), acyl CoA oxidase (AOX), acetylcholinesterase (AChE) and individual antioxidants (CAT, GST, GR, GPx and TGSH) were determined by specific spectrophotometric assays (Bocchetti and Regoli, 2006); TOSC toward peroxyl and hydroxyl radicals by gas chromatographic analyses (Regoli and Winston, 1999); lysosomal membrane stability by neutral red retention time (Lowe and Pipe, 1994); loss of DNA integrity by the comet assay and frequency of micronuclei (Venier et al, 1997;Machella et al, 2006). Data were analyzed by two-way analyses of variance (ANOVA) and principal component analyses (PCA) to test differences between sampling periods and species.…”