Vandegehuchte Michiel B, Kyndt Tina, Vanholme Bartel, Haegeman Annelies, Gheysen Godelieve, Janssen Colin R
Laboratory of Environmental Toxicology and Aquatic Ecology, Ghent University, J. Plateaustraat 22, B-9000 Ghent, Belgium.
Environ Int. 2009 May;35(4):700-6. doi: 10.1016/j.envint.2009.01.002. Epub 2009 Feb 26.
Most of the research on the epigenetic phenomenon of DNA methylation has been performed with vertebrates and plants. Knowledge on DNA methylation in Daphnia magna, a key test organism in aquatic toxicology, is completely lacking. Through epigenetic inheritance, effects of transient chemical exposure could be transferred to non-exposed generations, which could have a major impact on ecological risk assessment procedures. In this study, we determined if CpG methylation occurs in D. magna and if this can be influenced by exposure to toxic substances. Homologs of human DNA methyltransferases DNMT1, DNMT2 and DNMT3A were found in the partially available D. magna genome. Using an optimized "Amplification of Intermethylated Sites (AIMS)" technique, two methylated fragments were discovered in D. magna DNA. No homology was found for these sequences. The methylation and the D. magna origin of the fragments were confirmed with Southern analysis. This optimized AIMS technique was then applied to DNA of D. magna which were exposed to 180 microg/L Cd for two generations. Exposure resulted in a significant decrease in reproduction. The same methylated fragments with the same band intensity were observed in DNA of both non-exposed and exposed daphnids. As such, it could not be demonstrated that Cd exposure altered DNA methylation. However, the presence of DNA methylation in D. magna shows that potentially epigenetic effects may occur in this species.
大多数关于DNA甲基化表观遗传现象的研究都是在脊椎动物和植物中进行的。对于大型溞（水生毒理学中的一种关键受试生物）的DNA甲基化情况，我们却完全缺乏了解。通过表观遗传，短暂的化学物质暴露所产生的影响可能会传递给未暴露的后代，这可能会对生态风险评估程序产生重大影响。在本研究中，我们确定了大型溞中是否发生了CpG甲基化，以及这种甲基化是否会受到有毒物质暴露的影响。在部分已知的大型溞基因组中发现了人类DNA甲基转移酶DNMT1、DNMT2和DNMT3A的同源物。使用优化后的“甲基化位点扩增（AIMS）”技术，在大型溞DNA中发现了两个甲基化片段。这些序列没有同源性。通过Southern分析证实了片段的甲基化以及它们源自大型溞。然后将这种优化后的AIMS技术应用于两代暴露于180微克/升镉的大型溞的DNA。暴露导致繁殖能力显著下降。在未暴露和暴露的大型溞的DNA中都观察到了相同强度条带的相同甲基化片段。因此，无法证明镉暴露会改变DNA甲基化。然而，大型溞中存在DNA甲基化表明该物种可能会出现表观遗传效应。
