Machella Nicola, Battino Maurizio, Pisanelli Barbara, Regoli Francesco
Istituto di Biologia e Genetica, Università Politecnica delle Marche, Ancona, Italy.
Environ Mol Mutagen. 2006 Oct;47(8):579-86. doi: 10.1002/em.20231.
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.
使用单细胞凝胶电泳（SCGE）试验进行的遗传毒性研究表明，海洋无脊椎动物中DNA链断裂（SBs）的基础水平高于海洋脊椎动物，且更具变异性。这种DNA损伤水平的升高归因于大量碱不稳定位点，这是无脊椎动物细胞中紧密包装的DNA的特征。为了研究改变SCGE方案是否能人为调节高水平的SBs，使用蛋白酶K（PK）消化并结合含有不同浓度EDTA的检测缓冲液，对地中海贻贝（Mytilus galloprovincialis）的血细胞进行了SCGE实验。此外，还测试了生育三烯酚（可溶性抗氧化剂）和金精三羧酸（ATA；Ca(2+)/Mg(2+)-依赖性核酸酶抑制剂）的作用。将贻贝细胞中的SBs水平与陆生软体动物（蜗牛Helix aspersa）和硬骨鱼（海鲈Dicentrarchus labrax）细胞中的SBs水平进行了比较。使用EDTA、生育三烯酚和ATA通过酚提取法分离的贻贝DNA的完整性，通过凝胶电泳进一步检测。在SCGE试验的细胞处理过程中，将EDTA与PK消化相结合，或使用生育三烯酚或ATA，可降低贻贝细胞中的高SBs水平。在SCGE试验中，蜗牛和海鲈的SBs水平较低，且这些水平不受方案修改的影响。向贻贝基因组DNA的酚提取中添加EDTA、生育三烯酚或ATA，也可减少DNA片段化的程度。这些结果表明，贻贝的内部液体可能通过氧化和/或酶介导的途径增加DNA SBs的基础水平。贻贝被广泛用作评估海洋污染物遗传毒性危害的指示物种。我们的数据表明，在评估这些物种的DNA损伤时，应仔细考虑SCGE方案。
