Wilding Craig S, Trikic Michael Z, Hingston Joanne L, Copplestone David, Janet Tawn E
Genetics Department, Westlakes Research Institute, Moor Row, Cumbria CA24 3JY, UK.
Mutat Res. 2006 Jan 31;603(1):56-63. doi: 10.1016/j.mrgentox.2005.10.011. Epub 2005 Dec 27.
The compost worm Eisenia fetida is routinely used in ecotoxicological studies. A standard assay to assess genetic damage in this species would be extremely valuable. Since mitochondrial DNA (mtDNA) is known to exhibit an increased mutation rate following exposure to ionising radiation we assessed the validity of a mtDNA-based assay for measuring increases in mutation rate in laboratory-irradiated compost worms. To this end the mutation frequency in the mtDNA of the compost worm E. fetida was quantified following in vivo gamma-irradiation of adult worms in three dose groups. Five adult worms exposed to 1.4 mGy/h for 55 days (total dose 1.85 Gy), five adult worms exposed to 8.5 mGy/h for 55 days (total dose 11.22 Gy) and five adult control worms were used to assess the effect of irradiation on mtDNA mutation induction. DNA samples extracted from irradiated adult worms were used in high-fidelity PCR of a 486 bp region of mtDNA spanning the ATPase 8 gene, chosen for its high spontaneous mutation rate. PCR products were cloned and sequenced to identify mutations, with 89-102 clones successfully sequenced per individual. A significant elevation in mtDNA mutation frequency (p=0.032) was seen in worms exposed at the higher dose rate (8.5 mGy/h, total dose 11.22 Gy; mutation frequency 27.98+/-4.85 x 10(-5)mutations/bp) in comparison to controls (mutation frequency 12.68+/-3.06 x 10(-5)mutations/bp), but no elevation in mutation frequency (p=0.764) was seen for the lower dose rate (1.4 mGy/h, total dose 1.85 Gy; mutation frequency 13.74+/-1.29 x 10(-5)mutations/bp) compared with controls. This indicates that although the technique has the potential to detect an elevation in mutation frequency, it does not have sufficient sensitivity at the doses likely to be encountered in environmental monitoring scenarios.
堆肥蚯蚓赤子爱胜蚓常用于生态毒理学研究。一种评估该物种遗传损伤的标准检测方法将极具价值。由于已知线粒体DNA（mtDNA）在受到电离辐射后突变率会增加，我们评估了一种基于mtDNA的检测方法在测量实验室辐照堆肥蚯蚓突变率增加方面的有效性。为此，对三个剂量组的成年蚯蚓进行体内γ射线辐照后，对赤子爱胜蚓mtDNA中的突变频率进行了量化。将五只成年蚯蚓暴露于1.4 mGy/h下55天（总剂量1.85 Gy），五只成年蚯蚓暴露于8.5 mGy/h下55天（总剂量11.22 Gy），并使用五只成年对照蚯蚓来评估辐照对mtDNA突变诱导的影响。从辐照成年蚯蚓中提取的DNA样本用于对跨越ATPase 8基因的486 bp mtDNA区域进行高保真PCR，选择该区域是因为其自发突变率高。对PCR产物进行克隆和测序以鉴定突变，每个个体成功测序89 - 102个克隆。与对照组（突变频率12.68±3.06×10⁻⁵突变/bp）相比，高剂量率（8.5 mGy/h，总剂量11.22 Gy；突变频率27.98±4.85×10⁻⁵突变/bp）暴露的蚯蚓中mtDNA突变频率显著升高（p = 0.032），但与对照组相比，低剂量率（1.4 mGy/h，总剂量1.85 Gy；突变频率13.74±1.29×10⁻⁵突变/bp）的突变频率没有升高（p = 0.764）。这表明尽管该技术有检测突变频率升高的潜力，但在环境监测场景中可能遇到的剂量下，它没有足够的灵敏度。
